β1 Integrin-dependent Cell Adhesion to EMILIN-1 Is Mediated by the gC1q Domain

Abstract

EMILIN-1 (Elastin MicrofibrilInterface Located ProteIN), the prototype of the EMILIN family, consists of a cysteine-rich domain (EMI domain) at the N terminus, an extended region with a high potential coiled-coil structure, a short collagenous stalk, and a self-interacting globular gC1q-l domain. EMILIN-1 is an adhesive extracellular matrix constituent associated with elastic fibers, detected also in the proximity of cell surfaces. To localize the cell attachment site(s), monoclonal antibodies (mAbs) against EMILIN-1 or the gC1q-1 domain were used to inhibit cell attachment to EMILIN-1. Thus, one mAb mapping to the gC1q-1 domain caused complete inhibition of cell attachment. EMILIN-1 and gC1q-1 displayed a comparable dose-dependent ability to promote cell adhesion. Adhesion kinetics was similar to that of fibronectin (FN), reaching the maximum level of attachment at 20 min, but in the absence of cations adhesion was negligible. The relative adhesion strength to detach 50% of the cells was similar for EMILIN-1 and gC1q-1 (250 -270 ؋ g) but lower than that for FN (> >500). Cell adhesion to EMILIN-1 or gC1q-1 was completely blocked by a function-blocking ␤ 1 integrin subunit mAb. In contrast, adhesion to the complement C1q component was totally unaffected. Among the various function-blocking mAbs against the ␣ integrin subunits only the anti-␣ 4 fully abrogated cell adhesion to gC1q-1 and up to 70% to EMILIN-1. Furthermore, only K562 cells transfected with the ␣ 4 integrin chain, but not wild type K562, were able to adhere to EMILIN-1 and were specifically inhibited by anti-␣ 4 function-blocking mAb. Finally, cells attached to EMILIN-1 or gC1q-1, compared with cells plated on FN or vitronectin, which appeared well spread out on the substrate with prominent stress fibers and focal contacts, were much smaller with wide ruffles and a different organization status of the actin cytoskeleton along the cell periphery. This pattern was in accord with the ability of EMILIN-1 to promote cell movement.

Cross Talk among TGF- Signaling Pathways, Integrins, and the Extracellular Matrix

“…A host of other proteins also associate with fibrillin, e.g., fibulins, microfibril-associated glycoproteins (MAGP-1 and -2), perlecan, versican, and emilin1 (Ono et al 2009). Emilin1 modulates extracellular pro-TGF-b processing, as noted above, and is a ligand for integrin a4b1 (Spessotto et al 2003). Biglycan and decorin are proteoglycans that inhibit active TGF-b, and they associate with other components of elastic fibers in close proximity to fibrillin and may regulate fibrillin-1 expression (Trask et al 2000;Reinboth et al 2002;Schaefer et al 2004).…”

EMILIN1 represents a major stromal element determining human trophoblast invasion of the uterine wall

“…EMILIN1 belongs to a new family of proteins of the ECM characterized by a unique arrangement of structural domains, including a unique cysteine-rich sequence of approximately 80 amino acids at the amino-terminus, the EMI domain, an ␣-helical domain with high probability for coiled-coil structure formation in the central part, and a region homologous to the globular domain of C1q (gC1q domain) at the carboxyl-terminal end (Doliana et al, 1999;. EMILIN1 is often observed in vivo closely adjacent to the surface of cells; it is recognized at its gC1q1 domain by the ␣4␤1 integrin and it is able to promote tumor cell migration (Spessotto et al, 2003a).…”

Emilin3 is required for notochord sheath integrity and interacts with Scube2 to regulate notochord-derived Hedgehog signals

“…Emilin3 contains the characteristic cysteine-rich EMI domain at the Nterminal end, followed by a region forming three coiled-coil structures at the C-terminal end (Schiavinato et al, 2012). At variance with the other EMILIN/multimerin proteins, Emilin3 is not expressed in the cardiovascular system and it lacks the C-terminal gC1q domain, which is involved in cell attachment and integrin binding (Spessotto et al, 2003;Danussi et al, 2011). Emilin3 expression during mouse development is particularly abundant in the perichondrium of developing bones (Leimeister et al, 2002;Doi et al, 2004;Schiavinato et al, 2012).…”