**3. Integrins as Receptors for CgA and CgA-Derived Peptides**

#### *3.1. The Interaction of CgA and CgA Fragments with Integrins*

The first evidence for a role of integrins as receptors for full-length CgA and vasostatin-1 comes from a study on wound healing in injured mice [19]. This study has shown that CgA and vasostatin-1, at nanomolar concentrations, selectively interact with the integrin αvβ6 (see Table 1), suggesting that both polypeptides are natural ligands of this integrin. The integrin αvβ6 is an epithelial-specific cell-surface receptor of vitronectin, tenascin, fibronectin, and also of the latency associated protein of TGFβ1 [62–64]. In general, αvβ6 recognizes a site consisting of an arginine-glycine-aspartate (RGD) motif, followed by the LXXL/I motif (RGDLXXL/I) [65,66]. The latter motif folds into one α-helical turn upon binding to the receptor [65–70]. Interestingly, a short CgA-derived peptide comprising the residues 39–63 (CgA39-63, FETLRGDERILSILRHQNLLKELQD) is sufficient for highaffinity binding and highly selective recognition of αvβ6 (Ki: 15.5 ± 3.2 nM, Table 1) [71]. This peptide exhibits a degenerate RGDLXXL/I motif, in which a glutamate residue (E46) is present in place of the leucine downstream of the RGD sequence (RGDEXXL). Interestingly, in this peptide both the RGD motif (CgA43-45) and the adjacent sequence (CgA46-63) are crucial for αvβ6-integrin binding affinity and selectivity, as suggested by the observation that the replacement of RGD with RGE abrogates integrin recognition (Table 1), and the deletion of even a part of the C-terminal sequence markedly reduces binding affinity and selectivity [19]. The molecular determinants of αvβ6 recognition by CgA39-63 have been elucidated by NMR, computational, and biochemical studies [71]. Homonuclear and heteronuclear multidimensional NMR analyses of this peptide in physiological conditions have shown that the region between residues E46 and K59 has an α-helical conformation, while the RGD motif is relatively flexible; the first three turns of the α-helix are amphipathic, with the hydrophilic aminoacid residues E46, R47, S50 on one side and the hydrophobic I48, L49, I51, L52 on the opposite side [71]. The propensity of CgA39-63 to form an α-helix is consistent with the results of a previous NMR study on CgA47-66, an antifungal CgAderived peptide, showing all-helical conformation in trifluoroethanol, an α-helix-promoting solvent [72]. Saturation transfer difference (STD) spectroscopy experiments, performed with the extracellular region of human αvβ6 and isotopically labeled (13C/15N) CgA39-63, have shown that the hydrophobic residues I48, L49, I51, and L52 of the α-helix display the strongest STD values (>75%) [71], suggesting that these aminoacids contribute to receptor binding. Molecular docking experiments led to a model of receptor-ligand interactions that is highly reminiscent of that proposed for the proTGFβ1/αvβ6 complex [71].

**Table 1.** Binding affinity of CgA-derived fragments for integrins.


<sup>a</sup> *Ki*, equilibrium dissociation constant of the competitor (mean ± SEM). The *Ki* values were determined by competitive binding assay using an isoDGR-peroxidase conjugate as a probe for the integrin binding site [71].

No binding of CgA39-63 has been observed to other integrins (such as α1β1, α6β4, α3β1, α9β1 α6β7, α5β1, αvβ3, αvβ5, and αvβ8) at low-nanomolar concentrations [19]. However, peptides of containing the CgA39-63 region could recognize the integrin αvβ3 and other integrins of the RGD-family when used at high concentrations in the low-micromolar range [19]. For example, competitive binding assays performed with purified integrins showed that peptide CgA39-63 can bind αvβ6 and αvβ3 with Ki values of 15.5 and 2192 nM, respectively (Table 1), indicating that this peptide can recognize both integrins, but with markedly different affinities [71].

#### *3.2. Role of CgA/Integrin Interactions in Wound Healing*

The αvβ6 integrin is barely expressed in normal adult tissues, whereas it is highly expressed during wound healing, tissue remodeling, and embryogenesis [73,74]. This integrin is involved in TGFβ1 maturation, it regulates the expression of matrix metalloproteases and modulates keratinocyte adhesion, proliferation, and migration in wound healing [19,62,64,75]. It is possible, therefore, that CgA and its fragments have also a role in the regulation of the wound healing process, by interacting with this integrin. According to this view, experimental data showed that local injection of recombinant CgA1-439, but not of a CgA1-439 mutant with RGE in place of RGD, can accelerate wound healing in mice [19]. Immunohistochemical analysis of skin tissue sections obtained from injured mice, showed that CgA, but not the RGE mutant, could induce keratinocyte proliferation and thickening of epidermis, suggesting that CgA can regulate the keratinocytes physiology and the process of wound healing through an RGD-dependent mechanism that likely involves the αvβ6-integrin. Interestingly, both CgA and αvβ6 are expressed in wound keratinocytes [19,76]. The fact that both ligand and receptor are expressed at injured sites lends further support to the hypothesis that the CgA/αvβ6 interaction may have a pathophysiological role in this process.

Regarding the integrin αvβ3, this cell-adhesion receptor is an important player in endothelial cell biology and angiogenesis [77,78]. Although it is unlikely that this integrin has a receptor function for the circulating CgA polypeptides, considering its micromolar affinity, significant ligand-receptor interactions can possibly occur at sites where CgA is produced and, therefore, where this protein is present at high concentrations, such as in the microenvironment of wound keratinocytes and neuroendocrine secretory cells or in the microenvironment of neuroendocrine tumors. Furthermore, this interaction might occur on αvβ3-positive endothelial cell after the interaction with other high-affinity binding sites, i.e., through a sort of ligand-passing mechanism.
