SED1/MFG-E8: a bi-motif protein that orchestrates diverse cellular interactions

Abstract

MFG-E8 was initially identified as a principle component of the Milk Fat Globule, a membrane-encased collection of proteins and triglycerides that bud from the apical surface of mammary epithelia during lactation. It has since been independently identified in many species and by many investigators and given a variety of names, including p47, lactadherin, rAGS, PAS6/7, and BA-46. The acronym SED1 was proposed to bring cohesion to this nomenclature based upon it being a Secreted protein that contains two distinct functional domains: an N-terminal domain with two EGF-repeats, the second of which has an integrin-binding RGD motif, and a C-terminal domain with two Discoidin/F5/8C domains that bind to anionic phospholipids and/or extracellular matrices. SED1/MFG-E8 is now known to participate in a wide variety of cellular interactions, including phagocytosis of apoptotic lymphocytes and other apoptotic cells, adhesion between sperm and the egg coat, repair of intestinal mucosa, mammary gland branching morphogenesis, angiogenesis, among others. This article will explore the various roles proposed for SED1/MFG-E8, as well as its provocative therapeutic potential.

1). Structural motifs of SED1/MFG-E8 and Del1

Both proteins contain Notch like-EGF repeats, the second of which possess an RGD integrin-binding motif, as well as two discoidin/F5/8 C domains that are able to bind phospholipid bilayers and/or extracellular glycosides via 2-3 hairpin loops projecting from the central barrel core. SED1/MFG-E8 and Del1 are believed to facilitate cell-matrix adhesion via RGD-dependent binding to cell surface α_vβ_3/5 integrin receptors, whereas the discoidin/F5/8C domain is also thought to bind to cell membranes via intercalation into the lipid bilayer. Alternatively, the discoidin/F5/8C domains may coordinate binding to extracellular glycoside substrates, similar to that seen in the sugar-binding discoidin domains. Del1 contains a third EGF repeat not found within SED1.

2). Models for SED1/MFGE-8 function during sperm-egg binding

A) SED1/MFG-E8 binding to gamete surfaces appears to be mediated by the F5/8C domains, rather than the EGF repeats. Consequently, the simplest model suggests that SED1/MFG-E8 could function as a monomer, with one F5/8C domain binding to the sperm membrane and the other binding to the zona pellucida. B) Alternatively, SED1/MFG-E8 may facilitate sperm-egg binding as a dimmer, or oligomer, due to anti-parallel pairing of the EGF repeats, similar to that reported for the EGF domains of Ep-CAM [Balzar et al., 2001]. Reprinted from Ensslin and Shur, 2003.

3). The loss of SED1/MFG-E8 is associated with reduced MAPK activation in myoepithelial cells of the developing mammary gland

A) Although the level of total MAPK protein is similar between wildtype and SED1-null mammary gland organoids, as assessed by immunoblotting, the activated form of MAPK (P-MAPK) is greatly diminished in SED1-null organoids. Activation of the 44 kDa MAPK is reduced by nearly 50%, whereas activation of the 42 kDa isoform is closer to normal levels. B) MAPK (brown stain) is present in both the luminal and myoepithelial compartments, but activated MAPK (black arrows) is confined primarily to the myoepithelial compartment, consistent with the expression of α_v integrins on myoepithelial cells. MAPK activation is greatly reduced in the epithelial compartment of SED1-null organoids (p<0.001). Bars=0.05 mm C) Most cells with activated MAPK colocalize with smooth muscle actin (SMA), indicative of myoepithelial cells (white arrows); whereas others lie adjacent or outside of the SMA reactivity (pink arrows). Bars=0.05 mm D) The addition of SED1/MFG-E8 to primary epithelial cell cultures leads to a transient activation of MAPK. E) Results suggest that SED1/MFG-E8 is secreted by myoepithelial and/or basally from luminal epithelial cells. The RGD motif within the second EGF repeat binds α_vβ_5/3 integrins on myoepithelial cells, whereas adhesion to luminal epithelial cells may be mediated by intercalation of the F5/8C domains into the phospholipid bilayers. Ligation of α_vβ_5/3 integrin induces MAPK activation in myoepithelial cells, leading to proliferation of the epithelial compartment, duct elongation and branching. Reprinted from Ensslin and Shur, 2007.

4). SED1/MFG-E8 is secreted in association with exosomes

A) SED1/MFG-E8 may be secreted as a soluble protein through classical mechanisms, however SED1/MFG-E8's association with microvesicles suggests it is also secreted through non-classical pathways including B) apocrine shedding, as occurs in the epididymis, and C) fusion of multivesicular bodies (MVB) with the apical plasma membrane. Both apocrine shedding and MVB fusion result in the release of microvesicles, called exosomes, into the extracellular milieu.