Transmembrane-truncated αvβ3 integrin retains high affinity for ligand binding: evidence for an ‘inside-out’ suppressor?

The integrin ␣ v ␤ 3 has been shown to exist in low and high affinity conformations. Activation to the high affinity state is thought to depend on the "switchblade-like" opening, from a low affinity bent conformation with a closed headpiece to an extended form of the integrin with an open headpiece. Activation has been shown to depend on separation of the cytoplasmic domains. How cytoplasmic domain separation is related to separation of the transmembrane domains is unknown, and the distance of separation of the transmembrane domains required for activation has not been defined. A constrained secreted form of ␣ v ␤ 3 was engineered that introduced a 50-Å separation of the integrin C-terminal tails of the extracellular domains of the ␣ v and ␤ 3 subunits. Receptor binding and recognition by ligand-induced binding state (LIBS) monoclonal antibodies demonstrated that the mutant receptor was locked into a low affinity state that was likely in a partially extended conformation but with a closed headpiece. In the presence of RGD peptide, the constrained receptor was able to fully extend, as determined by full exposure of LIBS epitopes. In the presence of the appropriate LIBS antibody, high affinity ligand binding of the constrained receptor was achieved. The results support the existence of transient intermediate activation states of secreted ␣ v ␤ 3 . Furthermore, these results with the secreted ␣ v ␤ 3 receptor support a model for the full-length membrane-bound form of ␣ v ␤ 3 , whereby a 50-Å lateral separation of the integrin ␣ v and ␤ 3 transmembrane domains would be sufficient to enforce the switchbladelike opening to the extended conformation but insufficient for full receptor activation.Integrins are heterodimeric type I transmembrane proteins consisting of a single ␣ and ␤ subunit that mediates their effects on development, homeostasis, vessel growth, inflammation, hemostasis, and neoplasia through dynamic linkages between the extracellular matrix and cytoskeleton (1). Structural determination using x-ray crystallography, nuclear magnetic resonance, electron microscopy, and support from mutagenesis studies have provided evidence of dynamic intramolecular conformational changes that modulate ligand affinity and signal propagation (1-7). A low affinity state is maintained by a "highly bent" conformation with the integrin headpiece, which contains the ligand binding site, oriented toward the cell membrane. An extensive interface between the headpiece and the legs is observed in the x-ray structure of ␣ v ␤ 3 , which reveals a bent conformation assumed to correspond to this low affinity state (3). In addition, the ␣ and ␤ subunit tail domains also form an interface (3). Ligand binding or cytoplasmic domain separation is associated with loss of the headpiece-tailpiece interaction resulting in a switchblade-like opening of the integrin headpiece away from the membrane with associated separation of the ␣ and ␤ legs and exposure of activation epitopes (5,7,8). The orientation of the headpiece away from the membrane ov...

Section: ␣ V ␤ 3 Integrin-alkaline Phosphatase Fusion Proteins Are Sesupporting

confidence: 59%

Section: Discussionmentioning

confidence: 99%

A 50-Å Separation of the Integrin αvβ3 Extracellular Domain C Termini Reveals an Intermediate Activation State

Gline

Cambier

Govaerts

et al. 2004

“…The importance of the cytoplasmic tails is supported further by the observations that a point mutation in ␤ 3 (D723R) caused the receptor to be constitutively active in CHO cells (Fig. 5), and deletion of both the ␣ V and ␤ 3 tails renders purified ␣ V ␤ 3 competent to bind adhesive ligands (54). The ␣ V and ␤ 3 tails are also necessary for outside-in signaling triggered by ligand binding to ␣ V ␤ 3 (26,38,55).…”

Section: Discussionmentioning

confidence: 62%

Mechanisms and Consequences of Affinity Modulation of Integrin αVβ3 Detected with a Novel Patch-engineered Monovalent Ligand

Pampori¹,

Hato²,

Stupack³

et al. 1999

156

153

Integrin ␣ V ␤ 3 mediates diverse responses in vascular cells, ranging from cell adhesion, migration, and proliferation to uptake of adenoviruses. However, the extent to which ␣ V ␤ 3 is regulated by changes in receptor conformation (affinity), receptor diffusion/clustering (avidity), or post-receptor events is unknown. Affinity regulation of the related integrin, ␣ IIb ␤ 3 , has been established using a monovalent ligand-mimetic antibody, PAC1 Fab. To determine the role of affinity modulation of ␣ V ␤ 3 , a novel monovalent ligand-mimetic antibody (WOW-1) was created by replacing the heavy chain hypervariable region 3 of PAC1 Fab with a single ␣ V integrin-binding domain from multivalent adenovirus penton base. Both WOW-1 Fab and penton base bound selectively to activated ␣ V ␤ 3 , but not to ␣ IIb ␤ 3 , in receptor and cell binding assays. ␣ V ␤ 3 affinity varied with the cell type. Unstimulated B-lymphoblastoid cells bound WOW-1 Fab poorly (apparent K d ‫؍‬ 2.4 M), but acute stimulation with phorbol 12-myristate 13-acetate increased receptor affinity >30-fold (K d ‫؍‬ 80 nM), with no change in receptor number. In contrast, ␣ V ␤ 3 in melanoma cells was constitutively active, but ligand binding could be suppressed by overexpression of ␤ 3 cytoplasmic tails. Up-regulation of ␣ V ␤ 3 affinity had functional consequences in that it increased cell adhesion and spreading and promoted adenovirus-mediated gene transfer. These studies establish that ␣ V ␤ 3 is subject to rapid regulated changes in affinity that influence the biological functions of this integrin.Integrins mediate cell adhesion and signaling during many developmental, physiological, and pathological processes (1-4). The ␤ 3 integrin family includes ␣ IIb ␤ 3 , often referred to as the fibrinogen receptor, and ␣ V ␤ 3 , the vitronectin receptor. ␣ IIb ␤ 3 is confined to megakaryocytes and platelets and is required for platelet aggregation through interactions with Arg-Gly-Asp (RGD)-containing adhesive ligands, including fibrinogen and von Willebrand factor (5). ␣ V ␤ 3 is more widely expressed in proliferating endothelial cells, arterial smooth muscle cells, osteoclasts, platelets, and certain subpopulations of leukocytes and tumor cells (6, 7). The list of cognate ligands for ␣ V ␤ 3 overlaps that for ␣ IIb ␤ 3 , but includes others, such as osteopontin, matrix metalloproteinase-2, and adenovirus penton base, which do not interact with ␣ IIb ␤ 3 (6, 8 -10). In the adult organism, ␣ V ␤ 3 has been implicated in processes ranging from wound healing to tumor angiogenesis (11), arterial restenosis (12), osteoporosis (13), tumor progression (14), and adenovirus internalization (8).One fundamental function of integrins is ligand binding, which in many cases is rapidly regulated by a process variously referred to as "integrin activation," "inside-out signaling," or "affinity/avidity modulation" (15-19). Integrin activation encompasses at least two events: 1) modulation of receptor affinity through conformational changes in the ␣␤ heterodimer and 2) modulation...

“…Ligands-The ligands fibronectin (24), vitronectin (25), and fibrinogen (26) were purified from human blood and biotinylated as described previously (27,28).…”

Section: Methodsmentioning

confidence: 99%

“…The chains migrated at the molecular mass predicted for the intact and transmembrane truncated recombinant chains (␣v full-length, 150 kDa; ␣v-⌬TM, 125 kDa; ␣IIb full-length, 145 kDa; ␤3 full-length, 105 kDa; ␤5 full-length, 100 kDa; ␤3-⌬TM, 85 kDa; ␤6-⌬TM, 90 kDa). Each integrin was biologically active and showed ligand binding and divalent cation requirements predicted from the literature and also as demonstrated independently by us (13,27,28,30).Recombinant ␣v␤6 expressed as a transmembrane truncated soluble receptor bound two distinct classes of phages in phage display panning experiments from a linear 12-mer library as determined from the sequences of more than 100 clones: those containing RGD sequences (51%) and those containing an XX-DLXXLX motif (27%). Some phages contained RGD and also continued with the motif as RGDLXXL (9%), whereas others displayed the sequence RGDL (38%); the remaining phages that were bound often contained DLXXL-related sequences.…”

mentioning

confidence: 97%

Definition of an Unexpected Ligand Recognition Motif for αvβ6 Integrin

Kraft¹,

Diefenbach²,

Mehta

et al. 1999

Self Cite

130

128

Integrin interactions with extracellular matrix proteins are mediated by brief oligopeptide recognition sequences, and synthetic peptides containing such sequences can inhibit integrin binding to the matrix. The RGD peptide motif is recognized by many integrins including ␣v␤6, a specific receptor for fibronectin thought to support epithelial cell proliferation during wound healing and carcinoma progression. We report here the discovery of an unexpected non-RGD recognition motif for integrin ␣v␤6. We compared the recognition profiles of recombinant ␣v␤6 and ␣v␤3 integrins by using phage display screening employing 7-mer and 12-mer peptide libraries. As predicted, phages binding strongly to ␣v␤3 contained ubiquitous RGD sequences. However, on ␣v␤6, in addition to RGD-containing phages, one-quarter of the population from the 12-mer library contained the distinctive consensus motif DLXXL. A synthetic DLXXL peptide, RTDLDSLRTYTL, selected from the phage sequences (clone-1) was a selective inhibitor of RGD-dependent ligand binding to ␣v␤6 in isolated receptor assays (IC 50 ‫؍‬ 20 nM), and in cell adhesion assays (IC 50 ‫؍‬ 50 M). DLXXL peptides were highly specific inhibitors of ␣v␤6-fibronectin interaction as synthetic scrambled or reversed DLXXL peptides were inactive. NH 2 -and COOH-terminal modifications of the flanking amino acids suggested that the preceding two and a single trailing amino acid were also involved in interaction with ␣v␤6. The DLXXL sequence is present in several matrix components and in the ␤ chain of many integrins. Although there is as yet no precise biological role known for DLXXL, it is clearly a specific inhibitory sequence for integrin ␣v␤6 which has been unrecognized previously.Integrins are a family of heterodimeric class I transmembrane receptors involved in numerous cell-matrix and cell-cell adhesion phenomena (1). They can be grouped roughly into three classes: the ␤1 series, which are ubiquitous receptors for extracellular matrix (2); the ␤2 series, which are activatable on leukocytes and are triggered during the inflammatory response (3); and the ␣v series, which bind and mediate the cell response to provisional extracellular matrices found during wound repair and other pathological processes (4).The integrins ␣5␤1 (5), ␣IIb␤3 (6), ␣8␤1 (7), ␣v␤1 (8), ␣v␤3(9), and ␣v␤6 (10) all bind the Arg-Gly-Asp-(RGD) peptide sequence in fibronectin, where it is presented in a constrained loop (11). Soluble RGD-containing peptides can inhibit the interaction of each of these integrins with fibronectin. However, to analyze the function of individual fibronectin receptors in a particular cellular environment it is useful to have more specific inhibitors, and a variety of inhibitory antibodies, modified peptides, and non-peptidic substances has been developed. However, as yet no inhibitor has been discovered specific for ␣v␤6. ␣v␤6 is a rare integrin, induced during repair processes in epithelia (10, 12). Its only known specificities are for fibronectin (10), where it can be the dominant receptor me...