Neugebauer JM , Kwon S , Kim HS , Donley N , Tilak A , Sopory S , Christian JL .

5T32DK007115-37 NIDDK NIH HHS , R01 HD037976 NICHD NIH HHS , R01HD037976 NICHD NIH HHS , T32 DK007115 NIDDK NIH HHS

	Fig. 1. The S1 and S2 site of BMP4 is cleaved in BMP4/7 heterodimers. (A) Schematic illustration of cleavage patterns of BMP4 and BMP7 homodimers and position of epitope tags. (B and C) Two-cell embryos were injected with RNA encoding BMP4 (500 pg), BMP4S2G (1,000 pg), BMP7 (500 pg), or BMP4 and BMP7 (250 pg each). Blastocele fluid was extracted from the same number of embryos in each experimental group at the gastrula stage and proteins were deglycosylated and resolved by SDS PAGE. Immunoblots were probed with antibodies specific for myc (B), Flag, or HA epitopes (C) as indicated below each gel. Bands corresponding to ligand dimers, or to ligand or prodomain monomers, are indicated to the right of each gel.
	Fig. 2. Sequential cleavage of proBMP4 is required for maximal activity of homodimeric but not heterodimeric ligands. (A) Schematic illustration of cleavage patterns and relative bioactivity of ligands generated from wild-type and cleavage mutant BMP precursor proteins. (B) RNA encoding BMP7 or wild type or cleavage mutant forms of BMP4 were injected alone (200 pg) or in combination (100 pg of each RNA) near the dorsal midline of four-cell embryos. DMZ and VMZ explants were isolated at stage 10 and pSMAD1 levels were analyzed by immunoblot. Blots were reprobed with actin as a loading control, with the exception of the right panel, in which case duplicate samples were rerun and probed with actin. Blots shown in each of the three panels were run on different days, using samples from independent experiments, and exposed for different time periods. Thus, results can only be compared within each panel. For each panel, the relative level of pSMAD1 induced by injection of BMP4 or BMP7 alone, normalized to actin, and reported as a fraction of that induced by the two coinjected RNAs is indicated below each lane. (C and D) Quantitation of relative pSMAD1 levels in three independent experiments (mean ± SD). Normalized pSMAD1 levels in embryos injected with BMP7 or BMP4S2G alone (C), or with BMP7 or BMP4S1G alone (D) were added together, divided by two, and reported as a fraction of pSMAD1 levels induced by the two coinjected RNAs. (E) BMP RNAs were injected along with β-galactosidase RNA into one animal pole blastomere of 8- to 16-cell embryos. Embryos were stained for β-galactosidase activity (red) at stage 11 and then assayed for expression of Xbra by in situ hybridization (purple) as illustrated. The spread of Xbra signal outside of cells that express the precursor proteins (marked by red) was scored as 0+ to 3+ as illustrated. A total of 30–50 embryos were injected with each RNA per experiment and results are pooled from a minimum of three experiments.
	Fig. 3. The prodomain of BMP7 is not sufficient for maximal heterodimer activity. (A) Schematic illustration of ligands generated from native and chimeric BMP precursor proteins. (B) BMP RNAs were injected individually (200 pg) or together (100 pg of each RNA) near the dorsal midline of four-cell embryos. DMZ and VMZ explants were isolated at stage 10 and pSMAD1 levels were analyzed by immunoblot. Duplicate samples were rerun and blots were probed with actin as a loading control. All lanes are from the same pSmad1 immunoblot, aligned following removal of intervening lanes (following the 2nd and 4th lanes) using Photoshop. The first four lanes of the actin immunoblot are from the same gel, with the order rearranged to match the upper samples using Photoshop. Noncontiguous lanes are marked by white bars. The last two lanes were reprobed from samples shown in Fig. 4B. The relative level of pSMAD1 induced by injection of BMP7-4 or BMP7 alone, normalized to actin and reported as a fraction of that induced by the two co-injected RNAs, is indicated below each lane. (C) Quantitation of relative pSMAD1 levels induced by BMP4 or BMP7–4 in three independent experiments (mean ± SD). (D) Normalized pSMAD1 levels in embryos injected with BMP7 or BMP7–4 alone were added together, divided by two, and reported as a fraction of pSMAD1 levels induced by the two coinjected RNAs. Data from three independent experiments (mean ± SD). (E) BMP RNAs were injected along with β-galactosidase RNA into one animal pole blastomere of 8- to 16-cell embryos. Embryos were stained for β-galactosidase activity at stage 11 and then assayed for expression of Xbra by in situ hybridization. The spread of Xbra signal outside of cells that express the precursor proteins was scored as 0+ to 3+ as illustrated. A total of 30–50 embryos were injected with each RNA per experiment and results are pooled from a minimum of three experiments. (F) Protein present in blastocele fluid from the same number of embryos in each group was deglycosylated, resolved by SDS/PAGE under reducing or nonreducing conditions as indicated, and immunoblots were probed with antibodies specific for the myc epitope in the ligand domain. Bands corresponding to each ligand dimer or monomer are indicated to the right of each gel. Asterisks indicate two bands that may correspond to partially misfolded heterodimers.
	Fig. 4. The prodomain of BMP4 is necessary and sufficient to generate stable heterodimers with enhanced activity. (A) Schematic illustration of ligands generated from native and chimeric BMP precursor proteins. (B) BMP RNAs were injected individually (200 pg) or together (100 pg of each RNA) near the dorsal midline of four-cell embryos. DMZ and VMZ explants were isolated at stage 10 and pSMAD1 levels were analyzed by immunoblot. Actin levels were analyzed in duplicate samples as a loading control. The relative level of pSMAD1 induced by injection of BMP4-7 or BMP4 alone, normalized to actin and reported as a fraction of that induced by the two co-injected RNAs, is indicated below each lane. All lanes are from the same pSmad1 immunoblot, aligned following removal of an intervening lane (following the 2nd lane, marked by white bar) using Photoshop. The pSmad1 data shown in Figs. 2B (Lower Right), 3B, and 4B is all from a single injection experiment, run on the same gel and thus the BMP4 and BMP7 control lanes are the same in all three blots. Actin loading controls for these lanes are from duplicate samples that were rerun on different gels. (C) Normalized pSMAD1 levels in embryos injected with BMP4–7 or BMP4 alone were added together, divided by two, and reported as a fraction of pSMAD1 levels induced by the two coinjected RNAs. Data are from three independent experiments (mean ± SD). (D) BMP RNAs were injected along with β-galactosidase RNA into one animal pole blastomere of 8- to 16-cell embryos. Embryos were stained for β-galactosidase activity at stage 11 and then assayed for expression of Xbra by in situ hybridization. The spread of Xbra signal outside of cells that express the precursor proteins was scored as 0+ to 3+ as illustrated. A total of 30–50 embryos were injected with each RNA per experiment and results are pooled from a minimum of three experiments. (E) Protein present in blastocele fluid from the same number of embryos in each group was deglycosylated, resolved by SDS/PAGE under reducing or nonreducing conditions as indicated, and immunoblots were probed with antibodies specific for the myc epitope in the ligand domain. Bands corresponding to each ligand dimer or monomer are indicated to the right of each gel. The arrow marks a nonspecific band due to cross-reaction of the myc antibody with PNGase.
	Fig. 5. BMP4 and BMP7 prodomains remain associated with mature BMP4/7 heterodimers. (A) HEK cells were transfected with cDNAs encoding epitope-tagged BMP4 or BMP7 precursor proteins alone or together. Mature ligand fragments were immunoprecipitated (IP) from the cell media using antibodies specific for Flag or myc tags as indicated above each lane, and prodomain fragments were detected by probing immunoblots of immunoprecipitates or total protein (input) with antibodies specific for the BMP7 prodomain or the HA-epitope as indicated to the right of each gel. The position of the S1 only (arrow) or S1 and S2 cleaved BMP4 prodomain (arrowhead) is indicated. In each panel, samples were run on the same gel. An intervening lane was removed using Photoshop (between lane 8 and 9, marked by white bar) in the upper Right panel. Results were reproduced in three independent experiments. (B) Schematic illustration of the final product generated by proteolytic maturation of BMP4/7 heterodimers.
	Fig. 6. Hypothetical model for the role of the prodomain in promoting homodimer and heterodimer activity. (A) One or more ECM proteins bind to the prodomain of BMP4 intracellularly and traffic with the precursor to the cell surface where BMP4 is sequentially cleaved in a membrane proximal compartment. The transient ECM protein/prodomain/mature ligand complex that forms after S1 cleavage ensures that mature BMP4 is deposited in the ECM coincident with S2 cleavage, thereby facilitating association of the homodimeric ligand with HSPGs or other ECM proteins that are required for ligand stability. (B) The BMP4 prodomain, unlike the BMP7 prodomain, is sufficient to enable stable heterodimers to form. Following cleavage, the BMP4 and BMP7 prodomains remain associated with the heterodimeric ligand. The BMP4 prodomain binds to one or more ECM components, thereby anchoring the signaling complex in the matrix and/or presenting the heterodimeric ligand to HSPGs or other binding partners that facilitate signaling.

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