Atypical interactions of integrin α _V β ₈ with pro-TGF-β1

Abstract

Integrins α V β 6 and α V β 8 are specialized for recognizing pro-TGF-β and activating its growth factor by releasing it from the latency imposed by its surrounding prodomain. The integrin α V β 8 is atypical among integrins in lacking sites in its cytoplasmic domain for binding to actin cytoskeleton adaptors. Here, we examine α V β 8 for atypical binding to pro-TGF-β1. In contrast to α V β 6 , α V β 8 has a constitutive extended-closed conformation, and binding to pro-TGF-β1 does not stabilize the open conformation of its headpiece. Although Mn 2+ potently activates other integrins and increases affinity of α V β 6 for pro-TGF-β1 25-to 55-fold, it increases α V β 8 affinity only 2-to 3-fold. This minimal effect correlates with the inability of Mn 2+ and pro-TGF-β1 to stabilize the open conformation of the α V β 8 headpiece. Moreover, α V β 8 was inhibited by high concentrations of Mn 2+ and was stimulated and inhibited at markedly different Ca 2+ concentrations than α V β 6 . These unusual characteristics are likely to be important in the still incompletely understood physiologic mechanisms that regulate α V β 8 binding to and activation of pro-TGF-β.integrins | allostery | pro-TGF-β1 I ntegrins are cell-surface adhesion molecules that mediate cellcell, cell-extracellular matrix, and cell-pathogen interactions. In mammals, 24 different integrins are formed by specific, noncovalent association of 18 α-subunits with 8 β-subunits (1-5). Almost all integrins link to the actin cytoskeleton through talin and kindlin-binding motifs in their β-subunit cytoplasmic domains and thus provide traction for cell migration. The only exceptions are integrin α V β 8 , which binds through its β 8 -subunit to differentially express in adenocarcinoma of the lung (DAL-1, also known as Band 4.1B), and integrin α 6 β 4 , which links through its β 4 -subunit to intermediate filaments (6).Here, we focus on the atypical integrin α V β 8 and compare it to integrin α V β 6 . Integrins α V β 6 and α V β 8 are specialized for binding to and activation of pro-TGF-β1 and -β3. The pro-TGF-βs are biosynthesized and stored in tissues as latent forms. The dimeric TGF-β growth factor is kept latent by noncovalent association with its dimeric prodomain, which in turn is linked noncovalently and through disulfide bonds to a "milieu molecule" that stores the latent complex in the extracellular matrix or on the cell surface for subsequent, integrin-dependent activation. Integrins bind to an RGD motif that is present in the prodomains of pro-TGF-β1 and -β3. However, integrin binding alone is not sufficient for latent TGF-β activation by α V β 6 ; force is also required. Experiments suggest that tensile force is transmitted from the cytoskeleton to the integrin, is resisted by anchorage of TGF-β1 in the extracellular matrix or on cell surfaces, and results in distortion of prodomain straitjacket elements that loosely surround the growth factor followed by release of the growth factor (7,8). In some systems, activation of TGF-β1 by α V β 8 is dependent on...

A systematic survey of conformational states in β1 and β4 integrins using negative-stain electron microscopy

“…However, recent EM-based studies suggested that this simple assumption may not hold true, at least for some integrins. For example, it was shown that αVβ8 integrin assumed a constitutively extended conformation, regardless of the affinity states toward its physiological ligand, latent TGF-β (Wang et al, 2017;Minagawa et al, 2014). Furthermore, Springer and colleagues reported that the α5β1 integrin ectodomain rarely assumed the acutely bent conformation even in its resting (i.e.…”

High integrin α V β 6 affinity reached by hybrid domain deletion slows ligand-binding on-rate

“…Integrins α 4 β 1 , α 5 β 1 , and α V β 6 all appear to have bent-closed, extended-closed, and extended-open conformations. This conclusion is directly demonstrated for α 5 β 1 and α V β 6 by EM (10,11) and may be inferred for α 4 β 1 by the effects of Fabs that stabilize the closed, open, and extended states on affinity of soluble α 4 β 1 fragments for ligands and α 4 β 1 -dependent cell adhesion to specific ligand (6). We therefore interpret the increase in α Values are mean ± SD for one measurement each with Fn3 7-10 , Fn3 8-10 , and Fn3 9-10 , with the exception of the headpiece in Mg 2+ , for which Fn3 7-10 and Fn3 9-10 results could not be fit, and the fit ± fitting error is shown for Fn3 8-10 .…”

“…Overall, these results reveal major differences in the way in which important structural components in integrins, including the transmembrane/cytoplasmic domains and lower legs, regulate ligand-binding affinity by the integrin head. Recent studies on integrin α V β 8 also revealed only a four-to fivefold difference in affinity among clasped and unclasped ectodomain and headpiece forms (11). However, α V β 8 exists only in the closed conformation (11,14) and therefore is not comparable to α V β 6 , α 4 β 1 , or α 5 β 1 , all of which have both closed and open conformations.…”

“…Recent studies on integrin α V β 8 also revealed only a four-to fivefold difference in affinity among clasped and unclasped ectodomain and headpiece forms (11). However, α V β 8 exists only in the closed conformation (11,14) and therefore is not comparable to α V β 6 , α 4 β 1 , or α 5 β 1 , all of which have both closed and open conformations. Both α V β 6 and α V β 8 are specialized for activation of TGF-β1; it would be interesting to determine whether differences from α 4 β 1 and α 5 β 1 in α V β 6 affinity regulation also hold for integrins α V β 1 , α V β 3 , and…”

“…Our results clearly demonstrate that when α V β 6 reaches a sufficiently high affinity for ligand, its ligand-binding on-rate decreases. We discuss these results in terms of the closed and open conformations of the α V β 6 βI domain, head, headpiece, and ectodomain, which have been variously observed in crystal structure and electron microscopy studies that have demonstrated the high affinity of the open state (7,11,17). We use concepts derived from measurements of the affinity intrinsic to integrin conformational states and the conformational equilibria between states (5,6).…”