TGF-β2 uses the concave surface of its extended finger region to bind betaglycan’s ZP domain via three residues specific to TGF-β and inhibin-α

Abstract

Edited by Norma M. Allewell Betaglycan (BG) is a membrane-bound co-receptor of the TGF-␤ family that selectively binds transforming growth factor-␤ (TGF-␤) isoforms and inhibin A (InhA) to enable temporal-spatial patterns of signaling essential for their functions in vivo. Here, using NMR titrations of methyl-labeled TGF-␤2 with BG's C-terminal binding domain, BG ZP-C , and surface plasmon resonance binding measurements with TGF-␤2 variants, we found that the BG ZP-C -binding site on TGF-␤2 is located on the inner surface of its extended finger region. Included in this binding site are Ile-92, Lys-97, and Glu-99, which are entirely or mostly specific to the TGF-␤ isoforms and the InhA ␣-subunit, but they are unconserved in other TGF-␤ family growth factors (GFs). In accord with the proposed specificity-determining role of these residues, BG bound bone morphogenetic protein 2 (BMP-2) weakly or not at all, and TGF-␤2 variants with the corresponding residues from BMP-2 bound BG ZP-C more weakly than corresponding alanine variants. The BG ZP-C -binding site on InhA previously was reported to be located on the outside of the extended finger region, yet at the same time to include Ser-112 and Lys-119, homologous to TGF-␤2 Ile-92 and Lys-97, on the inside of the fingers. Therefore, it is likely that both TGF-␤2 and InhA bind BG ZP-C through a site on the inside of their extended finger regions. Overall, these results identify the BG ZP-C -binding site on TGF-␤2 and shed light on the specificity of BG for select TGF-␤-type GFs and the mechanisms by which BG influences their signaling.Betaglycan (BG) 3 is a co-receptor of the highly diversified transforming growth factor ␤ family (TGF-␤) of signaling proteins, which have essential roles in diverse processes, ranging from patterning of embryos and organs in embryogenesis to regulation of the endocrine and adaptive immune systems in adults (1-3). BG is expressed by epithelial, mesenchymal, and other cell types, but unlike the type I and type II signaling receptors (4 -6) that bind TGF-␤ family growth factors (GFs) to transduce the signal by phosphorylating Smads and other effectors (7-9), it is not required for signal transduction (10). BG is nonetheless essential in vertebrates, such as mice, where knockout of the BG gene is embryonic lethal (11).BG is a membrane-anchored proteoglycan composed of a large extracellular domain (766 amino acids in humans), a single transmembrane-spanning helix, and a short (43 amino acids in humans) highly-conserved cytoplasmic tail (Fig. 1A). BG's extracellular domain is composed of two subdomains, each roughly equal in size: the membrane-distal orphan domain (BG O ) and the C-terminal membrane-proximal zona pellucida domain (BG ZP ). The structure of BG's orphan domain is not known, but it is likely two tandem ␤-sandwiches, designated BG OR1 and BG OR2 , similar to that of endoglin (12), a homologous co-receptor of the TGF-␤ family with the same overall domain structure (13). BG ZP is likely composed of tandem immunoglobulin-like domains, d...

Betaglycan (TβRIII) is a Key Factor in TGF-β2 Signaling in Prepubertal Rat Sertoli Cells

“…TGF-βs also bind with high affinity to the co-receptors, betaglycan (TβRIII, BG) or endoglin [16], which may influence their function. In contrast to TGF-β1 and TGF-β3 which require only TβRI and TβRII to signal, TGF-β2 has a low affinity for TβRII, but binds with high affinity to betaglycan [17]: this binding mediates the interaction of TGF-β2 with TβRII and activates the signaling cascade for this ligand [18]. Thus, the abundance and localization of betaglycan is a key determinant of TGF-β pathway function and signaling outcomes.…”

Structural Adaptation in Its Orphan Domain Engenders Betaglycan with an Alternate Mode of Growth Factor Binding Relative to Endoglin

“…structures of BG ZP-C from rat (Lin et al, 2011) and mouse (Diestel et al, 2013) and homology to the ENG ZP (ENG ZP ) domain, whose structure was also recently reported (Saito et al, 2017). Through biochemical studies, rat BG, which has 5 cysteines within its orphan domain and 10 in its ZP domain, is found on cells as a monomer (Lopez-Casillas et al, 1993) and binds TGF-b homodimers asymmetrically with an overall 1:1 stoichiometry (Villarreal et al, 2016) using both its orphan domain and the C-terminal portion of its ZP domain, BG ZP-C (Henen et al, 2019;Mendoza et al, 2009;Villarreal et al, 2016) (Figure 1A). Human ENG (hENG), on the other hand, which has 5 cysteines in its orphan domain and 11 in its ZP domain, is found on cells as a disulfide-linked dimer and binds BMP-9 and BMP-10 using only its orphan domain (Alt et al, 2012).…”

“…The membrane-anchored co-receptors of the TGF-b family, BG and ENG, have essential roles potentiating the signaling activity of TGF-b2 and inhA and BMP-9 and BMP-10, respectively (Lewis et al, 2000;Lopez-Casillas et al, 1993;Nickel et al, 2018;Scharpfenecker et al, 2007). The selectivity with which BG and ENG recognize these GFs (Castonguay et al, 2011;Henen et al, 2019), together with the restricted pattern of their expression, is vital in enabling unique temporal-spatial patterns of signaling that underlie the unique functions of these GFs in vivo-for TGF-b2, morphogenetic transformation of endothelial progenitors in the developing heart and liver (Brown et al, 1999;Stenvers et al, 2003), for inhA antagonism of activin A and B in the anterior pituitary (Bernard et al, 2002;Lewis et al, 2000), and for endoglin activation of BMP-9 and BMP-10 signaling on endothelial cells required for normal development and maintenance of the vasculature (Roman and Hinck, 2017).…”

Structural biology of betaglycan and endoglin, membrane-bound co-receptors of the TGF-beta family

“…In 2012, Zhu et al showed that the extended N-terminus of the inhibin a-subunit is essential for inhibiting ActRIB binding, though whether this does so by blocking ActRIB binding at the type I site that lies at both a/b subunit interfaces, or only one of the a/b interfaces, was unclear. Importantly, the identification of the ZP-C binding on the underside of the fingers described above 65 provides an explanation for blocking of type I receptor binding at least at one of the a/b interfaces as this binding site overlaps extensively with that of the type I receptor site, which for all known type I receptors of the family, includes residues both from the underside of the fingers and from the heel helix of the adjoining monomer. It is therefore conceivable that binding of type I receptors to inhibin is blocked at one of a/b interfaces by the extended N-terminus of the a-subunit and the other by the betaglycan ZP-C domain.…”

“…Boxed residues highlight those that are either entirely (Lys 97 ) or mostly unique (Val 92 /Ile 92 and Glu 99 ) to TGF-bs and Inh a. Figure is adapted and reproduced with permission from Henen et al 65 (A color version of this figure is available in the online journal.) connects the orphan and ZP domains (A Hinck, unpublished observation), the latter being similar to the challenge encountered with full-length endoglin.…”