G-protein coupled receptor BAI3 promotes myoblast fusion in vertebrates Noumeira Hamouda,b, Viviane Trana,c, Louis-Philippe Croteaua,b, Artur Kaniaa,b,d,e, and Jean-François Côtéa,b,c,d,e,1 aInstitut de Recherches Cliniques de Montréal, Montreal, QC, Canada H2W 1R7; bProgrammes de Biologie Moléculaire, Département de Médecine and cDépartement de Biochimie, Université de Montréal, Montreal, QC, Canada H3T 1J4; and dDivision of Experimental Medicine and eDepartments of Biology and Anatomy and Cell Biology, McGill University, Montreal, QC, Canada H3A 1A3 Edited by Margaret Buckingham, Pasteur Institute, Paris, France, and approved January 24, 2014 (received for review July 25, 2013) Muscle fibers form as a result of myoblast fusion, yet the cell demonstrated that the myoblast fusion function of DOCK1 surface receptors regulating this process are unknown in verte- (ortholog of MBC) is conserved from flies to mice (22). Extending brates. In Drosophila, myoblast fusion involves the activation of this observation, conditional inactivation of Rac1 or N-WASP in the Rac pathway by the guanine nucleotide exchange factor Myo- developing mouse myoblasts was shown to impair fusion, further blast City and its scaffolding protein ELMO, downstream of cell- supporting an essential and evolutionarily conserved role for surface cell-adhesion receptors. We previously showed that the these genes in the fusion process (23, 24). However, mammalian mammalian ortholog of Myoblast City, DOCK1, functions in an evo- orthologs of fly profusion receptors, Nephrin and Neph proteins, lutionarily conserved manner to promote myoblast fusion in mice. In do not appear to be key regulators of primary myoblast fusion in search for regulators of myoblast fusion, we identified the G-protein mice (25, 26). Myomaker, or Transmembrane protein 8C, was coupled receptor brain-specific angiogenesis inhibitor (BAI3) as a cell recently shown to be essential for myoblast fusion, but its precise surface protein that interacts with ELMO. In cultured cells, BAI3 or function remains to be defined (27). A number of cell adhesion ELMO1/2 loss of function severely impaired myoblast fusion without proteins and promigratory receptors, including Integrin β1, affecting differentiation and cannot be rescued by reexpression of Talin1/2, CDON/Brother of CDON and M-/N-Cadherins are BAI3 mutants deficient in ELMO binding. The related BAI protein implicated in myoblast fusion in cell culture models, and in family member, BAI1, is functionally distinct from BAI3, because it vivo studies also support a contribution of Integrin β1and cannot rescue the myoblast fusion defects caused by the loss of BAI3 Talin1/2 to fusion (28–32). Recently, activation of the brain- function. Finally, embryonic muscle precursor expression of a BAI3 specific angiogenesis inhibitor 1 (BAI1) receptor by apoptotic cells mutant unable to bind ELMO was sufficient to block myoblast fusion residing in regenerating muscles was shown to promote myoblast in vivo. Collectively, our findings provide a role for BAI3 in the relay fusion in muscle repair (33). Thus, the canonical cell surface of extracellular fusion signals to their intracellular effectors, identi- receptors that control vertebrate primary myoblast fusion through fying it as an essential transmembrane protein for embryonic verte- – brate myoblast fusion. the conserved DOCK1 Rac pathway have remained elusive. BAI proteins belong to the cell adhesion subfamily of G-protein myotube formation | myogenesis | model system | ced-12 | RhoGTP coupled receptors (GPCRs) and are characterized by long ex- tracellular domains (ECDs) and intracellular domains (ICDs) (34). BAI3 regulates synapse density by binding to C1q-like he identity of molecular networks that control embryonic proteins, a family of small secreted proteins highly expressed in myogenesis and adult muscle regeneration is being unraveled T the brain, via its Thrombospondin repeats (TSRs) (34). BAI1 (1). The intricacy of such networks reflects the complicated cascade recruits ELMO/DOCK1 and acts as an apoptotic cell clearance of events underlying muscle development: myoblasts and satellite receptor by engaging phosphatidylserine exposed on the outer cells are first specified from precursors, then migrate to a precise leaflet of dying cells via its TSRs (35). Recent data demonstrated BIOLOGY location where they proliferate and fuse, and finally differentiate into postmitotic myofibers (2). Myoblast fusion is a critical step for DEVELOPMENTAL Significance the formation of embryonic muscle fibers, determines muscle size, and controls adult muscle regeneration (2), but its initiation re- mains poorly understood in vertebrates (3). Myoblast fusion is essential for multinucleated muscle fiber Dro- Genetic studies in Drosophila were instrumental in identifying formation. Regulators of fusion have been identified in sophila the molecules that control myoblast fusion by relaying signals , but the molecular mechanisms controlling vertebrate from the cell surface to the actin cytoskeleton (2). Cell adhesion myoblast fusion are poorly understood. We describe here receptors are expressed on two genetically distinguishable cell previously unidentified in vivo functional evidence that the populations of founder and fusion-competent myoblasts and are G-protein coupled receptor brain-specific angiogenesis in- hibitor (BAI3) is a receptor controlling myoblast fusion in critical to promote myoblast fusion (2). Dumbfounded/Kin-of-IrreC developing vertebrate muscles. We show that BAI3 binds the and Roughest/Irregular-optic-chiasma-C are expressed on the intracellular effector of myoblast fusion ELMO/DOCK1 and that founder myoblasts and associate with Stick and Stones and this binding is obligatory for fusion. Using in vivo manipulations Hibris found at the surface of fusion-competent myoblasts to – in the chicken embryo, we demonstrate that BAI3 binding to trigger intracellular signaling cascades leading to fusion (4 7). ELMO is essential for myoblast fusion. These experiments The signaling intermediates include scaffold proteins (ELMO, – identify BAI3 as a long-sought-after receptor controlling verte- Blown Fuse, Antisocial) (8 10), kinase (p21-activated kinase) brate myoblast fusion. (11), guanine nucleotide exchange factors (GEFs) [Myoblast City (MBC), Loner] (12, 13), GTPase (Rac), (14) and actin nucleation Author contributions: J.-F.C. designed research; N.H., V.T., and L.-P.C. performed research; regulators (Kette, WAVE, WASP, WASP-interacting protein) N.H., V.T., L.-P.C., A.K., and J.-F.C. analyzed data; and A.K. and J.-F.C. wrote the paper. (15–19). Some of these proteins ultimately promote the formation The authors declare no conflict of interest. of actin-driven podosome-like structures in the fusion-competent This article is a PNAS Direct Submission. cells that invade founder cells for membrane fusion (20, 21). 1To whom correspondence should be addressed. E-mail: [email protected]. These findings raise the question of the identity of in vivo This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. regulators of myoblast fusion in vertebrates. Previously, we 1073/pnas.1313886111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1313886111 PNAS | March 11, 2014 | vol. 111 | no. 10 | 3745–3750 Downloaded by guest on September 27, 2021 the presence of a GPCR–Autoproteolysis Inducing domain A 7 TM 1462 1509 (GAIN) in ECDs of some cell adhesion GPCRs that is involved TSR in autoproteolysis (36). In cancer cells, the BAI1 ECD is shed RGD 12345HBD GAIN and cleaved into smaller fragments that display antiangiogenic BAI1 properties (37). 1387 1563 Here, we present evidence for the identity of a transmembrane 1406 1452 protein that orchestrates myoblast fusion by relaying cell surface TSR 7 TM signals to the underlying cytoskeleton. We identify the GPCR 2345 HBD GAIN BAI3 as an interactor of ELMO/DOCK1 in myoblasts, whose BAI3 down-regulation in cultured myoblasts abolishes fusion. Rescue 1312 1499 experiments demonstrate that BAI3 engages the ELMO/DOCK1 pathway to mediate myoblast fusion. Finally, we show that in- C B B42 LexA terfering in vivo with BAI3’s ability to interact with ELMO im- Time (h) 0 24 48 72 +Leu paired primary myoblast fusion. Collectively, these results identify DOCK1 -Leu BAI3ICD ELMO11-727 BAI3 as an indispensable protein for myoblast fusion in vivo. ELMO1 ELMO2 BAI3ICD ELMO11-495 Results BAI2 ICD 532-727 GPCR BAI3, an ELMO-Binding Receptor, Is Expressed in Myoblasts. We BAI3 BAI3 ELMO1 reasoned that a myoblast cell surface receptor that binds directly E 1399-1513 1399-1458 to the ELMO/DOCK1 GEF module might relay an extracellular D 1399-1522 BAI1 BAI2 BAI3 signal to the cytoskeleton during fusion in vertebrates. We car- GST BAI3 BAI3 BAI3 Myc-ELMO1 + +++ GST ried out a yeast two-hybrid screen to identify proteins that in- Myc-ELMO1 + +++ 75 teract with ELMO1 and identified two potential candidate 75 transmembrane receptors. One of them is the previously repor- 50 ted ELMO-interacting BAI1 GPCR (35), and the other was the 25 Myc-ELMO1 related receptor BAI3 (Fig. 1A). Identity conservation between F 3OMLE-cyM G individual BAI protein family members is ∼40% and largely 727-31 restricted to the TSRs, GAIN domain, seven transmembrane 532-727 212-727 1-495 1 1-675 1-727 α 1-707 Myc-ELMO2 domains, and a short C-terminal region predicted to be -helical Myc-ELMO1 75 (38). Notably, BAI1 is characterized by five TSRs, whereas BAI2/3 75 GST-BAI3 have only four and additionally lack an Arg-Gly-Asp (RGD) motif GST-BAI3 50 A found exclusively on BAI1 (Fig. 1 ). Our yeast two-hybrid screen 75 75 suggests that ELMO1-interacting domains of BAI proteins are in GST α 50 GST the conserved predicted -helical region at the C terminus (Fig. TCL 5% A A 75 1 and Fig. S1 ). 75 Semiquantitative RT-PCR assays were performed to establish TCL 5% which DOCK, ELMO, and BAI members are expressed in C2C12, 50 a myoblast differentiation and fusion model cell line. In these, we detected constant expression levels of DOCK1 and DOCK4 mRNAs, whereas DOCK5 mRNA levels increased upon differen- Fig.