The Role of the Fibronectin IGD Motif in Stimulating Fibroblast Migration

Christopher J Millard,Ian R Ellis,Andrew R Pickford,Ana M Schor,Seth L Schor,Iain D Campbell

doi:10.1074/jbc.m707532200

Abstract

The motogenic activity of migration-stimulating factor, a truncated isoform of fibronectin (FN), has been attributed to the IGD motifs present in its FN type 1 modules. The structure-function relationship of various recombinant IGD-containing FN fragments is now investigated. Their structure is assessed by solution state NMR and their motogenic ability tested on fibroblasts. Even conservative mutations in the IGD motif are inactive or have severely reduced potency, while the structure remains essentially the same. A fragment with two IGD motifs is 100 times more active than a fragment with one and up to 10(6) times more than synthetic tetrapeptides. The wide range of potency in different contexts is discussed in terms of cryptic FN sites and cooperativity. These results give new insight into the stimulation of fibroblast migration by IGD motifs in FN.

Highlights

Fibronectin (FN)2 is a multifunctional, multidomain adhesive glycoprotein that plays a prominent role in wound healing, embryogenesis, and hemostasis [1]
Exposure of human dermal fibroblasts to migration-stimulating factor (MSF) provokes a change in phenotype, causing cells to migrate into three-dimensional gels of native type I collagen [5]
The RGD sequence has been shown to bind integrins and promote cell adhesion and fibril formation, the exact binding partner for the IGD motif is unknown, but its ability to promote cell migration, which is abolished by antibody to ␣v␤3 [8], suggests a possible interaction with integrins

Summary

IGD Motifs in Fibronectin and Cell Migration

Might be initiated through integrin cell attachment at the N terminus via a binding site other than the RGD region. We set out to explore the structure-function relationships of the IGD sequence in the context of intact Fn1 domains. Of particular interest are the third and fourth IGD motifs in MSF, one in the seventh Fn1 (in this report these modules will be described using a superscript nomenclature: 7Fn1, etc.), and one in 9Fn1. The effects of site-specific mutations on migration and structure are examined using fibroblast migration assays and solution state NMR. Both IGD motifs are shown to have significant migration-stimulating activity while contained within fully folded fibronectin modules

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

Relative potency of IGD motifs in different contexts