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Binding of integrin α1 to bone morphogenetic protein receptor IA suggests a novel role of integrin α1β1 in bone morphogenetic protein 2 signalling
Author’s Accepted Manuscript Binding of integrin α1 to bone morphogenetic protein receptor IA suggests a novel role of integrin α1β1 in bone morphogenetic protein 2 signalling Yan Zu, Xudong Liang, Jing Du, Shuai Zhou, Chun Yang www.elsevier.com
PII: DOI: Reference:
S0021-9290(15)00521-7 http://dx.doi.org/10.1016/j.jbiomech.2015.09.027 BM7335
To appear in: Journal of Biomechanics Received date: 21 May 2015 Revised date: 17 September 2015 Accepted date: 24 September 2015 Cite this article as: Yan Zu, Xudong Liang, Jing Du, Shuai Zhou and Chun Yang, Binding of integrin α1 to bone morphogenetic protein receptor IA suggests a novel role of integrin α1β1 in bone morphogenetic protein 2 signalling, Journal of Biomechanics, http://dx.doi.org/10.1016/j.jbiomech.2015.09.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Binding of integrin α1 to bone morphogenetic protein receptor IA suggests a novel role of integrin α1β1 in bone morphogenetic protein 2 signalling Yan Zu a, Xudong Liang a, b, Jing Du a, Shuai Zhoua and Chun Yanga,*
a
Institute of Biomechanics and Medical Engineering, School of Aerospace Engineering, Tsinghua
University, Beijing 100084, P.R. China b
Department of Mechanical and Aerospace Engineering, University of California, San Diego, La Jolla,
California 92093, USA
*To whom correspondence may be addressed: Chun Yang, Institute of Biomechanics and Medical Engineering, School of Aerospace Engineering, Tsinghua University, Beijing 100084, P.R. China. Tel.: 86-10-62788113; Fax: 86-10-62370855; E-mail: [email protected].
Keywords: bone morphogenetic protein receptor IA, integrin α1β1, steered molecular dynamics simulation
Manuscript type: Short Communications Word count (Introduction through Discussion): 1995
1
Abstract Here, we observed that integrin α1β1 and bone morphogenetic protein receptor (BMPR) IA formed a complex and co-localised in several cell types. However, the molecular interaction between these two molecules was not studied in detail to date and the role of the interaction in BMPR signalling remains unknown; thus, these were investigated here. In a steered molecular dynamics (SMD) simulation, the observed development of the rupture force related to the displacement between the A-domain of integrin α1 and the extracellular domain of BMPR IA indicated a strong molecular interaction within the integrin-BMPR complex. Analysis of the intermolecular forces revealed that hydrogen bonds, rather than salt bridges, are the major contributors to these intermolecular interactions. By using Enzyme-linked immunosorbent assay (ELISA) and co-immunoprecipitation (co-IP) experiments with site-directed mutants, we found that residues 85–89 in BMPR IA play the most important role for BMPR IA binding to integrin α1β1. These residues are the same as those responsible for bone morphogenetic protein 2 (BMP-2)/BMPR IA binding. In our experiments, we also found that the interference of integrin α1 up regulated the level of phosphorylated Smad1, 5, 8 which is the downstream of BMP/BMPR signalling. Therefore, our results suggest that integrin α1β1/BMPR IA may block BMP-2/BMPR IA complex information and interfere with the BMP-2 signalling pathway in cells.
Introduction The bone morphogenetic protein (BMP) signalling pathway plays important roles in osteoblast function and differentiation (Bragdon et al., 2011; Kamiya and Mishina, 2011). There are two types of BMP receptors (BMPRs): type I and type II receptors (Marie et al., 2014). Both receptors share a similar structure and comprise a short extracellular domain, a single trans-membrane domain, and an intracellular domain with serine-threonine kinase activity. The short extracellular domain is the binding site of BMPs. By binding to preformed oligomeric receptor complexes composed of BMPR I and BMPR II, BMP leads to an activation of the Smad signalling pathway; alternatively, BMP-2 can bind to the high-affinity BMPR IA and then recruit BMPR II into a hetero-oligomeric complex, which leads to 2
activation of the p38 pathway (Nohe et al., 2004; Shah et al., 2012) . BMPRsintensively interact with extracellular and membrane molecules that act as regulators of the BMP pathway. Among the regulating approaches of BMP/BMPR signalling (Wang et al., 2014), an emerging pattern of crosstalk and/or competition between BMP/BMPR and other pathways has received intensive attention (Bertrand et al., 2012; Wijk et al., 2009). Exploration of the proteins/pathways that are involved in crosstalk with BMP/BMPR pathways may further the current understanding of BMP regulation. Integrins, a large family of plasma membrane proteins with different α and β subunits, are involved in the processes of cell-cell and cell-extracellular matrix adhesion and signal transduction. Integrin triggers a cascade of signalling pathways which are closely associated with osteogenic cell differentiation, bone formation and repair (Marie et al., 2014). Among the integrin family members, α1β1, an important receptor for collagen I, collagen IV, and laminin, has been detected in various types of cells, and plays an essential role in the regulation of bone cells functions (Chiu et al., 2014). The A-domain (also known as the I-domain) in the α1 subunit provides a series of major binding sites for a variety of proteins (Calderwood et al., 1997). Lai et al. (2005) found that certain integrins, including αvβ1, αvβ3, αvβ5, αvβ8, co-localized with BMPRs in human U-2 OS osteosarcoma cells. Yang et al. (2014) demonstrated that integrin β5 co-localized with BMPR IA in human bone marrow mesenchymal stem cells (hBMMSCs). Having taken the previously reported results regarding certain integrins and BMPRs into consideration, we ask whether α1 subunit can co-localise and interact with BMPR IA via the A-domain. We first showed the co-localization of BMPR IA and integrin α1. Then we predicted the possible binding sites of BMPR IA and integrin α1 by SMD. Based on the SMD results, we further verified the mutant residues 85-89 roles by co-IP and ELISA. Together with RNA interference of integrin α1, we suggest that the integrin α1β1/BMPR IA complex may block the formation of the BMP-2/BMPR IA complex and interfere with the BMP-2 signalling pathway.
Material and Methods
Steered molecular dynamics (SMD) simulation of integrin-BMPR complex To study the detailed interactions between the two proteins, the integrin-BMPR docking complex 3
was equilibrated and subjected to an SMD-based simulation. In the simulation, constant pulling velocity was applied using the NAMD program and CHARMM22 force field. The backbone carbon atoms in residue 242 of integrin (integrin-R242) and residue 105 in BMPR (BMPR-R105) served as the fixed and pulling atoms, respectively. The pulling velocity was applied along the z-axis to detach the protein complex. Full details are included in the online supplemental materials. ELISA Plate 20 μg/ml integrin (7064-AB, R&D) overnight at 37 degrees onto 96-well plates (Maxisorp; Nunc). After washing with PBST, 4% BSA was added into the wells and incubated for 1 h at room temperature, and 10 μg/ml wt or mut BMPR IA recombinant protein (the detailed method for protein expression is included in the online supplemental materials) were incubated for 4 hours at room temperature. After washing three times, the wells were incubated with BMPR-IA antibody (sc20736, Santa) and then washed and incubated with goat Anti-Rabbit IgG H&L-Biotin (ab6720, abcam). The wells were incubated with streptavidin-conjugated horseradish peroxidase (Sigma-Aldrich) for 1 h at 4 °C. Finally, TMB (Sigma-Aldrich) reaction was used to visualise the interaction between integrin and wt or mut BMPR IA. Full methods are included in the online supplemental materials.
Results
BMPR IA co-localises with integrin α1β1 We compared the spatial distribution of BMPR IA with that of integrin α1β1 in MC-3T3 cells, primary osteoblasts, and BMMSCs using confocal analysis and immunoelectron microscopy. The immunostained and immunocolloidal gold labelled cells revealed that BMPR IA co-localised with integrin α1β1 (Fig. 1a, b). The association between BMPR IA and integrin α1β1 was confirmed using immunoprecipitation with specific antibodies against BMPR IA, followed by western blotting for integrin α1β1 (Fig. 1c). Integrin α1/BMPR IA complex formation, and the response to constant-velocity pulling 4
The simulated complex of integrin α1/BMPR IA was positioned in a water box with Na+ and Cl(Fig. 2a, Fig. S1) after scored by ZDOCK server (Supplementary Table S1, S2). In an SMD based simulation, a constant pulling velocity (0.005 Å/ps) was applied for 10 ns until the protein interfaces were completely separated (Fig. 2b). Figure 2c shows the evolution of the force and displacement during the simulation, as well as snapshots of the protein complex during the process. The rupture force represented the strength of the bonding between integrin α1 and BMPR IA during the first 2 ns, which was stronger than the non-bonding Van der Waals interaction; this suggested that the protein complex could establish a bond that produced a strong force during complex detachment. After the first 2 ns, the bonding mainly relied on the amino acid chain in the terminus of BMPR, and the two molecules underwent random contacts (snapshots 3 and 5). The random motion of the chain in the solvent led directly to oscillation of the force after 4 ns. In addition, the reallocated complex interface rebuilt some new bonds and produced a rupture force of 250 pN (see snapshot 4). Thus, the SMD simulation of integrin-BMPR complex demonstrated that a strong bond existed between the two proteins in the initial protein configuration and was rebuilt during the process of detachment, which proved that BMPR IA interacted with integrin α1β1. Identification of the binding domains Hydrogen bonds and salt bridges are known to play a central role in interface interaction through electrostatic complementarity (Kotzsch et al., 2008a; Xu et al., 1997). Figure 3a showed that only three interfacial salt bridges were found in the 10 ns simulation: D89-R287, R129-E293, and R129-E297 (BMPR residues given first). Figure 3b showed that about 30 hydrogen bonds were observed in the interface at the initial stage, each with a binding affinity of up to 4.5 kcal/mol and the change in the number of hydrogen bonds corresponded to the evolution of force. The components of binding sites with a bonding time over 10% of the simulation time were R85-89, R94-101, and R129 (Table 1; named domain I, II, and III, respectively). Considering that the number and formation time of salt bridges are unable to provide the rupture force observed in the simulation of up to 350 pN, hydrogen bonding was believed to be the major contributor to integrin-BMPR binding for the binding strength provided by hydrogen bonds was sufficient to produce the observed rupture force. 5
Numerical mutation of the binding domains SMD was performed on domain I and II mutants of BMPR IA, whereas domain III was neglected because of the single binding site (residue 129) and its random position in the terminal chain. As shown in Figure 4a, the force evolution for the domain I mutant by replacing polar and charged amino acids with alanine differed from that of the non-mutated domain as the peak of force disappeared in the first 2 ns and the highest rupture force decreased to 250 pN (i.e. less than normal). The mutation in domain II did not change the binding strength. As shown in Figure 4b, the number of hydrogen bonds in the interface decreased to 25 and 15 (the original number of hydrogen bonds for wt BMPR is 30), respectively. The increased reduction hydrogen bonds in the domain II mutant indicated that domain II provided more binding sites for hydrogen bond formation than domain I. However, these hydrogen bonds in domain II did not contribute to the rupture force between integrin α1 and BMPR. Experimental verification of the simulated binding domains Considering the SMD simulation, domain I was a candidate for integrin and BMPR binding sites because its numerical mutant showed a significant difference in the force evolution with only five binding sites decreasing. We examined exogenously expressed protein interactions through transfection of CHO cells with either HA-tagged wt or mut BMPR IA (Fig. 5a-c). The exogenous mut BMPR IA pulled down much less integrin than the endogenous wt BMPR IA (Fig. 5d). This indicated that the interaction with integrin was disrupted with mut BMPR IA. We then examined the interaction between wt or mut BMPR IA recombinant protein and integrin with ELISA. The result revealed that after mutating residues 85-89 the protein-protein interaction was significantly reduced (Fig. 5e). Hence, we suggested that the simulated binding domains were the actual binding sites of integrin α1-BMPR IA. As residues 85–89 in BMPR IA are the same as those responsible for bone morphogenetic protein 2 (BMP-2)/BMPR IA binding (Kotzsch et al., 2008b), we examined the crosstalk between BMP/BMPR and integrin signalling and found that the interference of integrin α1 up regulated the level of phosphorylated Smad1, 5, 8 which is the downstream of BMP/BMPR signaling (Fig. 5f).
6
Discussion BMPR IA, which phosphorylates Smad in the BMP signalling pathway, was suggested to form a complex with different integrins (Lai et al., 2005; Yang et al., 2014). Thus, we proposed that a functional relationship existed between these two signaling pathways. In the present study, Whe co-localization of BMPR IA and integrin α1was first demonstrated by immunocytochemistry and immunocolloidal gold labelling. According to the SMD analysis, residues 85–89 of BMPR IA were found to form the binding site to integrin α1β1. Together with experimentally demonstration by co-IP and ELISA, the binding sites of integrin α1β1/BMPR IA were found to be similar with the reported BMP2/BMPR binding sites. And indeed, we found that siRNA interference with integrin α1 up regulated the level of phosphorylated Smad 1, 5, 8 which are activated second messengers in BMP/BMPR signaling (Fig. 5f). Generally, the binding affinity of BMPs to type I receptors is higher than that to type II receptors (Cai et al., 2012). The A domain of integrin α1, studied in our work, is thought to be the main domain by which cells interact with the extracellular matrix. Thus, we chose the A-domain of integrin α1 and the extracellular domain of BMPR IA to study the interaction between the two molecules. However, other domains of integrin α1, other subunits of integrin, and other BMPRs should be studied to gain a complete understanding of the crosstalk between integrin and the BMP/BMPR pathways. Our study highlights the interaction between BMPR IA and integrin α1, and demonstrates that residues 85–89 of BMPR IA form the binding site to both integrin α1β1 and BMP-2. Yang et al. (2014) showed that nano-topography promotes co-localisation of integrin β5 and BMP2 receptors; therefore, research into how co-localisation of integrin and BMPR is regulated will be important for gaining a deeper understanding of the role played by the complex. Moreover, clarification of the regulation and downstream effects of the interaction between BMPR IA and integrin α1 may also uncover novel mechanisms by which these proteins could be targeted for therapeutic benefits.
Conflict of interest statement The authors declare that there are no conflicts of interest. 7
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (No. 31170885, 31400799, and 31370939) and Tsinghua University (2011Z02175 and 2012Z02133). We would like to thank Editage (www.editage.com) for English language editing.The study sponsors had no role in study design; collection, analysis and interpretation of data; writing the manuscript; or the decision to submit the manuscript for publication.
References Bertrand, F. E., Angus, C. W., Partis, W. J., Sigounas, G., 2012. Developmental pathways in colon cancer: crosstalk between WNT, BMP, Hedgehog and Notch. Cell Cycle 11, 4344-4351. Bragdon, B., Moseychuk, O., Saldanha, S., King, D., Julian, J., Nohe, A., 2011. Bone morphogenetic proteins: a critical review. Cellular Signalling 23, 609-620. Cai, J., Pardali, E., Sanchez-Duffhues, G., ten Dijke, P., 2012. BMP signaling in vascular diseases. FEBS letters 586, 1993-2002. Calderwood, D.A., Tuckwell, D.S., Eble, J., Kühn, K., Humphries, M.J., 1997. The integrin α1 A-domain is a ligand binding site for collagens and laminin. Journal of Biological Chemistry 272, 12311-12317. Chiu, L.-H., Lai, W.-F.T., Chang, S.-F., Wong, C.-C., Fan, C.-Y., Fang, C.-L., Tsai, Y.-H., 2014. The effect of type II collagen on MSC osteogenic differentiation and bone defect repair. Biomaterials 35, 2680-2691. Du, J., Chen, X., Liang, X., Zhang, G., Xu, J., He, L., Zhan, Q., Feng, X.-Q., Chien, S., Yang, C., 2011. Integrin activation and internalization on soft ECM as a mechanism of induction of stem cell differentiation by ECM elasticity. Proceedings of the National Academy of Sciences of the USA 108, 9466-9471. Hu, R., Liu, W., Li, H., Yang, L., Chen, C., Xia, Z.-Y., Guo, L.-J., Xie, H., Zhou, H.-D., Wu, X.-P., 2011. A Runx2/miR-3960/miR-2861 regulatory feedback loop during mouse osteoblast differentiation. Journal of Biological Chemistry 286, 12328-12339. 8
Kamiya, N., Mishina, Y., 2011. New insights on the roles of BMP signaling in bone—a review of recent mouse genetic studies. Biofactors 37, 75-82. Kotzsch, A., Nickel, J., Seher, A., Heinecke, K., van Geersdaele, L., Herrmann, T., Sebald, W., Mueller, T.D., 2008a. Structure analysis of bone morphogenetic protein-2 type I receptor complexes reveals a mechanism of receptor inactivation in juvenile polyposis syndrome. Journal of Biological Chemistry 283, 5876-5887. Lai, C.F., Cheng, S.L., 2005. αvβ integrins play an essential role in BMPϋ2 induction of osteoblast differentiation. Journal of Bone and Mineral Research 20, 330-340. Lee, K.-S., Kim, H.-J., Li, Q.-L., Chi, X.-Z., Ueta, C., Komori, T., Wozney, J.M., Kim, E.-G., Choi, J.-Y., Ryoo, H.-M., 2000. Runx2 is a common target of transforming growth factor β1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Molecular and Cellular Biology 20, 8783-8792. Liu, F., Pouponnot, C., Massagué, J., 1997. Dual role of the Smad4/DPC4 tumor suppressor in TGFβ-inducible transcriptional complexes. Genes & Development 11, 3157-3167. Marie, P.J., Haÿ, E., Saidak, Z., 2014. Integrin and cadherin signaling in bone: role and potential therapeutic targets. Trends in Endocrinology & Metabolism 25, 567-575. Nohe, A., Keating, E., Knaus, P., Petersen, N.O., 2004. Signal transduction of bone morphogenetic protein receptors. Cellular Signalling 16, 291-299. Shah, P., Keppler, L., Rutkowski, J., 2012. Bone morphogenic protein: An elixir for bone grafting-a review. Journal of Oral Implantology 38, 767-778. Tang, D.-Q., Cao, L.-Z., Burkhardt, B.R., Xia, C.-Q., Litherland, S.A., Atkinson, M.A., Yang, L.-J., 2004. In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow. Diabetes 53, 1721-1732. Ten Dijke, P., Yamashita, H., Sampath, T.K., Reddi, A.H., Estevez, M., Riddle, D.L., Ichijo, H., Heldin, C.-H., Miyazono, K., 1994. Identification of type I receptors for osteogenic protein-1 and bone morphogenetic protein-4. Journal of Biological Chemistry 269, 16985-16988. 9
Tokuyasu, K., 1989. Use of poly (vinylpyrrolidone) and poly (vinyl alcohol) for cryoultramicrotomy. The Histochemical Journal 21, 163-171. van Wijk B., van den Berg G., Abu-Issa R., Barnett P., van der Velden S., Schmidt M., Ruijter J.M., Kirby M.L., Moorman A.F., van den Hoff M.J., 2009. Epicardium and myocardium separate from a common precursor pool by crosstalk between bone morphogenetic protein–and fibroblast growth factor–signaling pathways. Circulation research 105, 431-441. Wang, R.N., Green, J., Wang, Z., Deng, Y., Qiao, M., Peabody, M., Zhang, Q., Ye, J., Yan, Z., Denduluri, S., Idowu, O., Li, M., Shen, C., Hu, A., Haydon, R.C., Kang, R., Mok, J., Lee, M.J., Luu, H.L., Shi, L.L., 2014. Bone morphogenetic protein (BMP) signaling in development and human diseases. Genes & Diseases 1, 87-105. Xu, D., Tsai, C.-J., Nussinov, R., 1997. Hydrogen bonds and salt bridges across protein-protein interfaces. Protein Engineering 10, 999-1012. Yang, J., McNamara, L.E., Gadegaard, N., Alakpa, E.V., Burgess, K.V., Meek, R.D., Dalby, M.J., 2014. Nanotopographical induction of osteogenesis through adhesion, bone morphogenic protein cosignaling, and regulation of microRNAs. ACS Nano 8, 9941-9953.
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Table 1. Hydrogen bonding and binding time of the integrin α1β1-BMPR IA complex. Binding sites
Residues
Binding time (>10%)
ASP89- ARG287
52.80%
ASP89- TYR156
50.20%
GLN86- GLU317
22.40%
CYS87- TYR156
14.20%
GLN86- TYR156
12.60%
PHE85- ASP159
8.20%
CYS101- TRP158
14.80%
GLU100- SER154
14.60%
PRO91- ARG287
14.20%
ILE99- TYR156
13.80%
GLN94- ASN289
13.80%
GLU100- GLN214
12.60%
GLN94- ASN289
12.20%
GLU100- ILE155
9.60%
GLN94- ASN286
8.40%
ARG129- GLU297
81.20%
ARG129- GLU293
59.80%
ARG129- SER291
17.20%
ARG129- SER291
14.80%
SER127- LYS294
9.00%
THR104- ILE212
8.80%
ARG103- ASP159
7.60%
Domain I
Domain II
Domain III
11
Figure Legends Figure 1. BMPR IA interacts with integrin α1β1 (a) Immunofluorescence of BMPR IA and integrin α1 shows their co-localisation in MC-3T3 cells, primary osteoblasts, and BMMSCs. (b) Double-immunogold labelling for integrin α1β1 (5-nm gold particles) and BMPR IA (10-nm gold particles). ~6 co-localizations per cell were observed. (c) BMPR IA-integrin α1β1 co-IP in cells transfected with increasing amounts of BMPR IA. Lysates were subjected to IP with HA antibody, followed by immunoblotting with anti-integrin α1β1. IgG was used as a non-specific antibody for immunoprecipitation.
Figure 2. Integrin α1β1-BMPR IA complex and the evolution of the force. (a) Schematic representation of the complex solvated in water and neutralised with Na+ and Cl-. The solvent is represented in light blue bulk and the proteins are described in ribbon, with dark blue for integrin and yellow for BMPR. (b) Dissociation of the integrin-BMPR complex in the SMD simulation. The backbone carbon atom of integrin-R242 is fixed, while a constant pulling velocity is applied along the z-axis to the backbone carbon atom of BMPR-R105. (c) During the first 2 ns, the force peaks at 300 pN and 350 pN, and the corresponding complex structure is visualised in snapshots 1 and 2. After the strong rupture force in the first 2 ns, the bonding relies on the amino acid chain in the terminus of BMPR and undergoes random contacts (snapshots 3 and 5). The reallocated complex interface rebuilds part of the protein bonds and produces a rupture force of 250 pN in snapshot 4.
Figure 3. Bonding distance and strength of integrin α1-BMPR IA complex. (a) Distance between the oxygen atoms of a salt bridge in the SMD simulation. Only three interfacial salt bridges were found in the 10-ns simulation. (b) The number of hydrogen bonds formed in the integrin-BMPR complex. The total number in the interface is approximately 30 at the initial stage. Points 1 and 2 represent two peaks of rupture force in the simulation, and they coincide with the drop in number of hydrogen bonds. In the 3rd stage, hydrogen bonds were formed and damaged continuously.
12
Figure 4. The force spectra and hydrogen bond number of the normal BMPR IA protein and its domain I and II mutants. (a) The domain I mutant showed altered force evolution in the SMD simulation. However, the domain II mutant presented a similar tendency in force evolution compared with that of the normal complex. (b) The hydrogen bond number decreases to 25 in the domain I mutation and 15 in the domain II mutation. This decrease is larger in the domain II mutation (up to 10) compared with that in the domain I mutation. The mutated BMPR also lacks the ability to reconstruct the hydrogen bond after 2 ns, which is in contrast to the normal situation.
Figure 5. The role of residuces 85-89 in BMPR/integrin complex formation and downstream BMP2/BMPR signaling. (a) The sequence of HA-tagged wt BMPR IA plasmid. (b) The sequence of HA-tagged mut BMPR IA plasmid. (c) Sequence alignment of HA-tagged wt and mut BMPR IA plasmid. (d) BMPR IA-integrin α1β1 co-IP in CHO cells transfected with HA-tagged wt BMPR IA or mut BMPR IA. Lysates were subjected to IP with HA antibody, followed by immunoblotting with anti-integrin α1β1. (e) Interaction between integrin and wt BMPR IA (grey) or mut BMPR IA (black) analysed by ELISA. P values indicate significant differences in the interaction levels (mean ± SEM; n = 4); ** p < 0.01, ***p<0.001. (f) p-Smad in the presence of integrin alpha1 siRNA or scramble e were analyzed by western blotting (mean ± SEM; n=3). ** P < 0.01, ***p<0.001. “NC”: negative control.
13
Figure
Figure2-v1
Figure3-v1
Figure4-v1
Figure5-v1
PII: DOI: Reference:
S0021-9290(15)00521-7 http://dx.doi.org/10.1016/j.jbiomech.2015.09.027 BM7335
To appear in: Journal of Biomechanics Received date: 21 May 2015 Revised date: 17 September 2015 Accepted date: 24 September 2015 Cite this article as: Yan Zu, Xudong Liang, Jing Du, Shuai Zhou and Chun Yang, Binding of integrin α1 to bone morphogenetic protein receptor IA suggests a novel role of integrin α1β1 in bone morphogenetic protein 2 signalling, Journal of Biomechanics, http://dx.doi.org/10.1016/j.jbiomech.2015.09.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Binding of integrin α1 to bone morphogenetic protein receptor IA suggests a novel role of integrin α1β1 in bone morphogenetic protein 2 signalling Yan Zu a, Xudong Liang a, b, Jing Du a, Shuai Zhoua and Chun Yanga,*
a
Institute of Biomechanics and Medical Engineering, School of Aerospace Engineering, Tsinghua
University, Beijing 100084, P.R. China b
Department of Mechanical and Aerospace Engineering, University of California, San Diego, La Jolla,
California 92093, USA
*To whom correspondence may be addressed: Chun Yang, Institute of Biomechanics and Medical Engineering, School of Aerospace Engineering, Tsinghua University, Beijing 100084, P.R. China. Tel.: 86-10-62788113; Fax: 86-10-62370855; E-mail: [email protected].
Keywords: bone morphogenetic protein receptor IA, integrin α1β1, steered molecular dynamics simulation
Manuscript type: Short Communications Word count (Introduction through Discussion): 1995
1
Abstract Here, we observed that integrin α1β1 and bone morphogenetic protein receptor (BMPR) IA formed a complex and co-localised in several cell types. However, the molecular interaction between these two molecules was not studied in detail to date and the role of the interaction in BMPR signalling remains unknown; thus, these were investigated here. In a steered molecular dynamics (SMD) simulation, the observed development of the rupture force related to the displacement between the A-domain of integrin α1 and the extracellular domain of BMPR IA indicated a strong molecular interaction within the integrin-BMPR complex. Analysis of the intermolecular forces revealed that hydrogen bonds, rather than salt bridges, are the major contributors to these intermolecular interactions. By using Enzyme-linked immunosorbent assay (ELISA) and co-immunoprecipitation (co-IP) experiments with site-directed mutants, we found that residues 85–89 in BMPR IA play the most important role for BMPR IA binding to integrin α1β1. These residues are the same as those responsible for bone morphogenetic protein 2 (BMP-2)/BMPR IA binding. In our experiments, we also found that the interference of integrin α1 up regulated the level of phosphorylated Smad1, 5, 8 which is the downstream of BMP/BMPR signalling. Therefore, our results suggest that integrin α1β1/BMPR IA may block BMP-2/BMPR IA complex information and interfere with the BMP-2 signalling pathway in cells.
Introduction The bone morphogenetic protein (BMP) signalling pathway plays important roles in osteoblast function and differentiation (Bragdon et al., 2011; Kamiya and Mishina, 2011). There are two types of BMP receptors (BMPRs): type I and type II receptors (Marie et al., 2014). Both receptors share a similar structure and comprise a short extracellular domain, a single trans-membrane domain, and an intracellular domain with serine-threonine kinase activity. The short extracellular domain is the binding site of BMPs. By binding to preformed oligomeric receptor complexes composed of BMPR I and BMPR II, BMP leads to an activation of the Smad signalling pathway; alternatively, BMP-2 can bind to the high-affinity BMPR IA and then recruit BMPR II into a hetero-oligomeric complex, which leads to 2
activation of the p38 pathway (Nohe et al., 2004; Shah et al., 2012) . BMPRsintensively interact with extracellular and membrane molecules that act as regulators of the BMP pathway. Among the regulating approaches of BMP/BMPR signalling (Wang et al., 2014), an emerging pattern of crosstalk and/or competition between BMP/BMPR and other pathways has received intensive attention (Bertrand et al., 2012; Wijk et al., 2009). Exploration of the proteins/pathways that are involved in crosstalk with BMP/BMPR pathways may further the current understanding of BMP regulation. Integrins, a large family of plasma membrane proteins with different α and β subunits, are involved in the processes of cell-cell and cell-extracellular matrix adhesion and signal transduction. Integrin triggers a cascade of signalling pathways which are closely associated with osteogenic cell differentiation, bone formation and repair (Marie et al., 2014). Among the integrin family members, α1β1, an important receptor for collagen I, collagen IV, and laminin, has been detected in various types of cells, and plays an essential role in the regulation of bone cells functions (Chiu et al., 2014). The A-domain (also known as the I-domain) in the α1 subunit provides a series of major binding sites for a variety of proteins (Calderwood et al., 1997). Lai et al. (2005) found that certain integrins, including αvβ1, αvβ3, αvβ5, αvβ8, co-localized with BMPRs in human U-2 OS osteosarcoma cells. Yang et al. (2014) demonstrated that integrin β5 co-localized with BMPR IA in human bone marrow mesenchymal stem cells (hBMMSCs). Having taken the previously reported results regarding certain integrins and BMPRs into consideration, we ask whether α1 subunit can co-localise and interact with BMPR IA via the A-domain. We first showed the co-localization of BMPR IA and integrin α1. Then we predicted the possible binding sites of BMPR IA and integrin α1 by SMD. Based on the SMD results, we further verified the mutant residues 85-89 roles by co-IP and ELISA. Together with RNA interference of integrin α1, we suggest that the integrin α1β1/BMPR IA complex may block the formation of the BMP-2/BMPR IA complex and interfere with the BMP-2 signalling pathway.
Material and Methods
Steered molecular dynamics (SMD) simulation of integrin-BMPR complex To study the detailed interactions between the two proteins, the integrin-BMPR docking complex 3
was equilibrated and subjected to an SMD-based simulation. In the simulation, constant pulling velocity was applied using the NAMD program and CHARMM22 force field. The backbone carbon atoms in residue 242 of integrin (integrin-R242) and residue 105 in BMPR (BMPR-R105) served as the fixed and pulling atoms, respectively. The pulling velocity was applied along the z-axis to detach the protein complex. Full details are included in the online supplemental materials. ELISA Plate 20 μg/ml integrin (7064-AB, R&D) overnight at 37 degrees onto 96-well plates (Maxisorp; Nunc). After washing with PBST, 4% BSA was added into the wells and incubated for 1 h at room temperature, and 10 μg/ml wt or mut BMPR IA recombinant protein (the detailed method for protein expression is included in the online supplemental materials) were incubated for 4 hours at room temperature. After washing three times, the wells were incubated with BMPR-IA antibody (sc20736, Santa) and then washed and incubated with goat Anti-Rabbit IgG H&L-Biotin (ab6720, abcam). The wells were incubated with streptavidin-conjugated horseradish peroxidase (Sigma-Aldrich) for 1 h at 4 °C. Finally, TMB (Sigma-Aldrich) reaction was used to visualise the interaction between integrin and wt or mut BMPR IA. Full methods are included in the online supplemental materials.
Results
BMPR IA co-localises with integrin α1β1 We compared the spatial distribution of BMPR IA with that of integrin α1β1 in MC-3T3 cells, primary osteoblasts, and BMMSCs using confocal analysis and immunoelectron microscopy. The immunostained and immunocolloidal gold labelled cells revealed that BMPR IA co-localised with integrin α1β1 (Fig. 1a, b). The association between BMPR IA and integrin α1β1 was confirmed using immunoprecipitation with specific antibodies against BMPR IA, followed by western blotting for integrin α1β1 (Fig. 1c). Integrin α1/BMPR IA complex formation, and the response to constant-velocity pulling 4
The simulated complex of integrin α1/BMPR IA was positioned in a water box with Na+ and Cl(Fig. 2a, Fig. S1) after scored by ZDOCK server (Supplementary Table S1, S2). In an SMD based simulation, a constant pulling velocity (0.005 Å/ps) was applied for 10 ns until the protein interfaces were completely separated (Fig. 2b). Figure 2c shows the evolution of the force and displacement during the simulation, as well as snapshots of the protein complex during the process. The rupture force represented the strength of the bonding between integrin α1 and BMPR IA during the first 2 ns, which was stronger than the non-bonding Van der Waals interaction; this suggested that the protein complex could establish a bond that produced a strong force during complex detachment. After the first 2 ns, the bonding mainly relied on the amino acid chain in the terminus of BMPR, and the two molecules underwent random contacts (snapshots 3 and 5). The random motion of the chain in the solvent led directly to oscillation of the force after 4 ns. In addition, the reallocated complex interface rebuilt some new bonds and produced a rupture force of 250 pN (see snapshot 4). Thus, the SMD simulation of integrin-BMPR complex demonstrated that a strong bond existed between the two proteins in the initial protein configuration and was rebuilt during the process of detachment, which proved that BMPR IA interacted with integrin α1β1. Identification of the binding domains Hydrogen bonds and salt bridges are known to play a central role in interface interaction through electrostatic complementarity (Kotzsch et al., 2008a; Xu et al., 1997). Figure 3a showed that only three interfacial salt bridges were found in the 10 ns simulation: D89-R287, R129-E293, and R129-E297 (BMPR residues given first). Figure 3b showed that about 30 hydrogen bonds were observed in the interface at the initial stage, each with a binding affinity of up to 4.5 kcal/mol and the change in the number of hydrogen bonds corresponded to the evolution of force. The components of binding sites with a bonding time over 10% of the simulation time were R85-89, R94-101, and R129 (Table 1; named domain I, II, and III, respectively). Considering that the number and formation time of salt bridges are unable to provide the rupture force observed in the simulation of up to 350 pN, hydrogen bonding was believed to be the major contributor to integrin-BMPR binding for the binding strength provided by hydrogen bonds was sufficient to produce the observed rupture force. 5
Numerical mutation of the binding domains SMD was performed on domain I and II mutants of BMPR IA, whereas domain III was neglected because of the single binding site (residue 129) and its random position in the terminal chain. As shown in Figure 4a, the force evolution for the domain I mutant by replacing polar and charged amino acids with alanine differed from that of the non-mutated domain as the peak of force disappeared in the first 2 ns and the highest rupture force decreased to 250 pN (i.e. less than normal). The mutation in domain II did not change the binding strength. As shown in Figure 4b, the number of hydrogen bonds in the interface decreased to 25 and 15 (the original number of hydrogen bonds for wt BMPR is 30), respectively. The increased reduction hydrogen bonds in the domain II mutant indicated that domain II provided more binding sites for hydrogen bond formation than domain I. However, these hydrogen bonds in domain II did not contribute to the rupture force between integrin α1 and BMPR. Experimental verification of the simulated binding domains Considering the SMD simulation, domain I was a candidate for integrin and BMPR binding sites because its numerical mutant showed a significant difference in the force evolution with only five binding sites decreasing. We examined exogenously expressed protein interactions through transfection of CHO cells with either HA-tagged wt or mut BMPR IA (Fig. 5a-c). The exogenous mut BMPR IA pulled down much less integrin than the endogenous wt BMPR IA (Fig. 5d). This indicated that the interaction with integrin was disrupted with mut BMPR IA. We then examined the interaction between wt or mut BMPR IA recombinant protein and integrin with ELISA. The result revealed that after mutating residues 85-89 the protein-protein interaction was significantly reduced (Fig. 5e). Hence, we suggested that the simulated binding domains were the actual binding sites of integrin α1-BMPR IA. As residues 85–89 in BMPR IA are the same as those responsible for bone morphogenetic protein 2 (BMP-2)/BMPR IA binding (Kotzsch et al., 2008b), we examined the crosstalk between BMP/BMPR and integrin signalling and found that the interference of integrin α1 up regulated the level of phosphorylated Smad1, 5, 8 which is the downstream of BMP/BMPR signaling (Fig. 5f).
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Discussion BMPR IA, which phosphorylates Smad in the BMP signalling pathway, was suggested to form a complex with different integrins (Lai et al., 2005; Yang et al., 2014). Thus, we proposed that a functional relationship existed between these two signaling pathways. In the present study, Whe co-localization of BMPR IA and integrin α1was first demonstrated by immunocytochemistry and immunocolloidal gold labelling. According to the SMD analysis, residues 85–89 of BMPR IA were found to form the binding site to integrin α1β1. Together with experimentally demonstration by co-IP and ELISA, the binding sites of integrin α1β1/BMPR IA were found to be similar with the reported BMP2/BMPR binding sites. And indeed, we found that siRNA interference with integrin α1 up regulated the level of phosphorylated Smad 1, 5, 8 which are activated second messengers in BMP/BMPR signaling (Fig. 5f). Generally, the binding affinity of BMPs to type I receptors is higher than that to type II receptors (Cai et al., 2012). The A domain of integrin α1, studied in our work, is thought to be the main domain by which cells interact with the extracellular matrix. Thus, we chose the A-domain of integrin α1 and the extracellular domain of BMPR IA to study the interaction between the two molecules. However, other domains of integrin α1, other subunits of integrin, and other BMPRs should be studied to gain a complete understanding of the crosstalk between integrin and the BMP/BMPR pathways. Our study highlights the interaction between BMPR IA and integrin α1, and demonstrates that residues 85–89 of BMPR IA form the binding site to both integrin α1β1 and BMP-2. Yang et al. (2014) showed that nano-topography promotes co-localisation of integrin β5 and BMP2 receptors; therefore, research into how co-localisation of integrin and BMPR is regulated will be important for gaining a deeper understanding of the role played by the complex. Moreover, clarification of the regulation and downstream effects of the interaction between BMPR IA and integrin α1 may also uncover novel mechanisms by which these proteins could be targeted for therapeutic benefits.
Conflict of interest statement The authors declare that there are no conflicts of interest. 7
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (No. 31170885, 31400799, and 31370939) and Tsinghua University (2011Z02175 and 2012Z02133). We would like to thank Editage (www.editage.com) for English language editing.The study sponsors had no role in study design; collection, analysis and interpretation of data; writing the manuscript; or the decision to submit the manuscript for publication.
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Kamiya, N., Mishina, Y., 2011. New insights on the roles of BMP signaling in bone—a review of recent mouse genetic studies. Biofactors 37, 75-82. Kotzsch, A., Nickel, J., Seher, A., Heinecke, K., van Geersdaele, L., Herrmann, T., Sebald, W., Mueller, T.D., 2008a. Structure analysis of bone morphogenetic protein-2 type I receptor complexes reveals a mechanism of receptor inactivation in juvenile polyposis syndrome. Journal of Biological Chemistry 283, 5876-5887. Lai, C.F., Cheng, S.L., 2005. αvβ integrins play an essential role in BMPϋ2 induction of osteoblast differentiation. Journal of Bone and Mineral Research 20, 330-340. Lee, K.-S., Kim, H.-J., Li, Q.-L., Chi, X.-Z., Ueta, C., Komori, T., Wozney, J.M., Kim, E.-G., Choi, J.-Y., Ryoo, H.-M., 2000. Runx2 is a common target of transforming growth factor β1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Molecular and Cellular Biology 20, 8783-8792. Liu, F., Pouponnot, C., Massagué, J., 1997. Dual role of the Smad4/DPC4 tumor suppressor in TGFβ-inducible transcriptional complexes. Genes & Development 11, 3157-3167. Marie, P.J., Haÿ, E., Saidak, Z., 2014. Integrin and cadherin signaling in bone: role and potential therapeutic targets. Trends in Endocrinology & Metabolism 25, 567-575. Nohe, A., Keating, E., Knaus, P., Petersen, N.O., 2004. Signal transduction of bone morphogenetic protein receptors. Cellular Signalling 16, 291-299. Shah, P., Keppler, L., Rutkowski, J., 2012. Bone morphogenic protein: An elixir for bone grafting-a review. Journal of Oral Implantology 38, 767-778. Tang, D.-Q., Cao, L.-Z., Burkhardt, B.R., Xia, C.-Q., Litherland, S.A., Atkinson, M.A., Yang, L.-J., 2004. In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow. Diabetes 53, 1721-1732. Ten Dijke, P., Yamashita, H., Sampath, T.K., Reddi, A.H., Estevez, M., Riddle, D.L., Ichijo, H., Heldin, C.-H., Miyazono, K., 1994. Identification of type I receptors for osteogenic protein-1 and bone morphogenetic protein-4. Journal of Biological Chemistry 269, 16985-16988. 9
Tokuyasu, K., 1989. Use of poly (vinylpyrrolidone) and poly (vinyl alcohol) for cryoultramicrotomy. The Histochemical Journal 21, 163-171. van Wijk B., van den Berg G., Abu-Issa R., Barnett P., van der Velden S., Schmidt M., Ruijter J.M., Kirby M.L., Moorman A.F., van den Hoff M.J., 2009. Epicardium and myocardium separate from a common precursor pool by crosstalk between bone morphogenetic protein–and fibroblast growth factor–signaling pathways. Circulation research 105, 431-441. Wang, R.N., Green, J., Wang, Z., Deng, Y., Qiao, M., Peabody, M., Zhang, Q., Ye, J., Yan, Z., Denduluri, S., Idowu, O., Li, M., Shen, C., Hu, A., Haydon, R.C., Kang, R., Mok, J., Lee, M.J., Luu, H.L., Shi, L.L., 2014. Bone morphogenetic protein (BMP) signaling in development and human diseases. Genes & Diseases 1, 87-105. Xu, D., Tsai, C.-J., Nussinov, R., 1997. Hydrogen bonds and salt bridges across protein-protein interfaces. Protein Engineering 10, 999-1012. Yang, J., McNamara, L.E., Gadegaard, N., Alakpa, E.V., Burgess, K.V., Meek, R.D., Dalby, M.J., 2014. Nanotopographical induction of osteogenesis through adhesion, bone morphogenic protein cosignaling, and regulation of microRNAs. ACS Nano 8, 9941-9953.
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Table 1. Hydrogen bonding and binding time of the integrin α1β1-BMPR IA complex. Binding sites
Residues
Binding time (>10%)
ASP89- ARG287
52.80%
ASP89- TYR156
50.20%
GLN86- GLU317
22.40%
CYS87- TYR156
14.20%
GLN86- TYR156
12.60%
PHE85- ASP159
8.20%
CYS101- TRP158
14.80%
GLU100- SER154
14.60%
PRO91- ARG287
14.20%
ILE99- TYR156
13.80%
GLN94- ASN289
13.80%
GLU100- GLN214
12.60%
GLN94- ASN289
12.20%
GLU100- ILE155
9.60%
GLN94- ASN286
8.40%
ARG129- GLU297
81.20%
ARG129- GLU293
59.80%
ARG129- SER291
17.20%
ARG129- SER291
14.80%
SER127- LYS294
9.00%
THR104- ILE212
8.80%
ARG103- ASP159
7.60%
Domain I
Domain II
Domain III
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Figure Legends Figure 1. BMPR IA interacts with integrin α1β1 (a) Immunofluorescence of BMPR IA and integrin α1 shows their co-localisation in MC-3T3 cells, primary osteoblasts, and BMMSCs. (b) Double-immunogold labelling for integrin α1β1 (5-nm gold particles) and BMPR IA (10-nm gold particles). ~6 co-localizations per cell were observed. (c) BMPR IA-integrin α1β1 co-IP in cells transfected with increasing amounts of BMPR IA. Lysates were subjected to IP with HA antibody, followed by immunoblotting with anti-integrin α1β1. IgG was used as a non-specific antibody for immunoprecipitation.
Figure 2. Integrin α1β1-BMPR IA complex and the evolution of the force. (a) Schematic representation of the complex solvated in water and neutralised with Na+ and Cl-. The solvent is represented in light blue bulk and the proteins are described in ribbon, with dark blue for integrin and yellow for BMPR. (b) Dissociation of the integrin-BMPR complex in the SMD simulation. The backbone carbon atom of integrin-R242 is fixed, while a constant pulling velocity is applied along the z-axis to the backbone carbon atom of BMPR-R105. (c) During the first 2 ns, the force peaks at 300 pN and 350 pN, and the corresponding complex structure is visualised in snapshots 1 and 2. After the strong rupture force in the first 2 ns, the bonding relies on the amino acid chain in the terminus of BMPR and undergoes random contacts (snapshots 3 and 5). The reallocated complex interface rebuilds part of the protein bonds and produces a rupture force of 250 pN in snapshot 4.
Figure 3. Bonding distance and strength of integrin α1-BMPR IA complex. (a) Distance between the oxygen atoms of a salt bridge in the SMD simulation. Only three interfacial salt bridges were found in the 10-ns simulation. (b) The number of hydrogen bonds formed in the integrin-BMPR complex. The total number in the interface is approximately 30 at the initial stage. Points 1 and 2 represent two peaks of rupture force in the simulation, and they coincide with the drop in number of hydrogen bonds. In the 3rd stage, hydrogen bonds were formed and damaged continuously.
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Figure 4. The force spectra and hydrogen bond number of the normal BMPR IA protein and its domain I and II mutants. (a) The domain I mutant showed altered force evolution in the SMD simulation. However, the domain II mutant presented a similar tendency in force evolution compared with that of the normal complex. (b) The hydrogen bond number decreases to 25 in the domain I mutation and 15 in the domain II mutation. This decrease is larger in the domain II mutation (up to 10) compared with that in the domain I mutation. The mutated BMPR also lacks the ability to reconstruct the hydrogen bond after 2 ns, which is in contrast to the normal situation.
Figure 5. The role of residuces 85-89 in BMPR/integrin complex formation and downstream BMP2/BMPR signaling. (a) The sequence of HA-tagged wt BMPR IA plasmid. (b) The sequence of HA-tagged mut BMPR IA plasmid. (c) Sequence alignment of HA-tagged wt and mut BMPR IA plasmid. (d) BMPR IA-integrin α1β1 co-IP in CHO cells transfected with HA-tagged wt BMPR IA or mut BMPR IA. Lysates were subjected to IP with HA antibody, followed by immunoblotting with anti-integrin α1β1. (e) Interaction between integrin and wt BMPR IA (grey) or mut BMPR IA (black) analysed by ELISA. P values indicate significant differences in the interaction levels (mean ± SEM; n = 4); ** p < 0.01, ***p<0.001. (f) p-Smad in the presence of integrin alpha1 siRNA or scramble e were analyzed by western blotting (mean ± SEM; n=3). ** P < 0.01, ***p<0.001. “NC”: negative control.
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