A Novel Cell Adhesion Region in Tropoelastin Mediates Attachment to Integrin αVβ5

Pearl Lee,Daniel V Bax,Marcela M.M Bilek,Anthony S Weiss

doi:10.1074/jbc.m113.518381

Abstract

Tropoelastin protein monomers assemble to form elastin. Cellular integrin αVβ3 binds RKRK at the C-terminal tail of tropoelastin. We probed cell interactions with tropoelastin by deleting the RKRK sequence to identify other cell-binding interactions within tropoelastin. We found a novel human dermal fibroblast attachment and spreading site on tropoelastin that is located centrally in the molecule. Inhibition studies demonstrated that this cell adhesion was not mediated by either elastin-binding protein or glycosaminoglycans. Cell interactions were divalent cation-dependent, indicating integrin dependence. Function-blocking monoclonal antibodies revealed that αV integrin(s) and integrin αVβ5 specifically were critical for cell adhesion to this part of tropoelastin. These data reveal a common αV integrin-binding theme for tropoelastin: αVβ3 at the C terminus and αVβ5 at the central region of tropoelastin. Each αV region contributes to fibroblast attachment and spreading, but they differ in their effects on cytoskeletal assembly.

Highlights

Cellular integrin ␣V␤3 binds to RKRK at the C-terminal tail of tropoelastin
human dermal fibroblast (HDF) Attachment and Spreading on N terminus to domain 18 (N18) and 17–27 Is Mediated via ␣V-containing Integrin(s)—Integrins are inhibited by chelation of cations, so we examined the effect of EDTA on cell binding to ⌬RKRK, N18, and 17–27 (Fig. 6A)
Previous studies investigating the binding of fibroblasts to tropoelastin have shown that integrin ␣V␤3 mediates cell binding to the C-terminal GRKRK sequence [12]

Summary

Background

Cellular integrin ␣V␤3 binds to RKRK at the C-terminal tail of tropoelastin. Results: Inhibition of integrin ␣V␤5 hinders cell adhesion to tropoelastin constructs comprising domains 17 and 18. The 67-kDa EBP is a peripheral membrane splice variant of ␤-galactosidase that complexes with the integral membrane proteins carboxypeptidase A and sialidase to form a transmembrane elastin receptor [14] This receptor binds XGXXPG consensus sequences and, in particular, VGVAPG within exon 24 of elastin [15] binding elicits cell responses (16 –20), it is not responsible for direct human dermal fibroblast (HDF)tropoelastin interactions; rather, EBP appears to function as a detector of elastin-derived fragments that are generated when elastin is damaged [10]. Integrin ␣V␤3 on HDFs was found to recognize the extreme C-terminal RKRK motif of human tropoelastin [12] This interaction does not account for the full cell-binding activity of tropoelastin. We have used recombinant tropoelastin constructs to identify a new cell-binding site in tropoelastin and to identify integrin ␣V␤5 as the major receptor for this novel region

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION