No abstract

Unlike most receptors, Notch serves as both the receiver and direct transducer of signaling events. Activation can be mediated by one of five membrane-bound ligands of either the Delta-like (-1, -2, -4) or Jagged/Serrate (-1, -2) families. Alternatively, dissociation of the Notch heterodimer with consequent activation can also be mediated experimentally by calcium chelators or by mutations that destabilize the Notch1 heterodimer, such as in the human disease T cell acute lymphoblastic leukemia. Here we show that MAGP-2, a protein present on microfibrils, can also interact with the EGF-like repeats of Notch1. Co-expression of MAGP-2 with Notch1 leads to both cell surface release of the Notch1 extracellular domain and subsequent activation of Notch signaling. Moreover, we demonstrate that the C-terminal domain of MAGP-2 is required for binding and activation of Notch1. Based on the high level of homology, we predicted and further showed that MAGP-1 can also bind to Notch1, cause the release of the extracellular domain, and activate signaling. Notch1 extracellular domain release induced by MAGP-2 is dependent on formation of the Notch1 heterodimer by a furinlike cleavage, but does not require the subsequent ADAM metalloprotease cleavage necessary for production of the Notch signaling fragment. Together these results demonstrate for the first time that the microfibrillar proteins MAGP-1 and MAGP-2 can function outside of their role in elastic fibers to activate a cellular signaling pathway.Notch signaling is best known for its role in cell fate determination and is critical for regulating multiple cellular processes in many different tissues, including those of the nervous, hematopoietic, and vascular systems (1). Controlled by membrane-tethered ligands on apposing cells, ligand binding initiates canonical Notch signaling and leads to the proteolytic release of the Notch intracellular domain (NICD).2 The NICD fragment travels to the nucleus, interacts with a DNA-binding protein CSL (CBF1/Su(H)/LAG-1), and activates expression of target genes such as HES1.At least three proteolytic events are required for Notch activation through CSL. The first cleavage is ligand-independent and occurs during maturation of the co-linear Notch protein. Either during trafficking or at the cell surface, Notch is cleaved by a furin-like convertase into two fragments, the extracellular domain (N EC ) and the transmembrane-anchored intracellular domain (N TM ), that remain associated through non-covalent interactions (2-4). This "heterodimer" is the predominant form of Notch on the plasma membrane and is required for ligand-induced CSL-dependent Notch signaling (4). Accordingly, ligand engagement by the heterodimeric Notch receptor leads to sequential ADAM and ␥-secretase cleavage events that facilitate the release of NICD from its membrane tether to activate signaling (5, 6).Underscoring the importance of Notch signaling is the finding that more than 50% of T-ALL patient samples tested so far carry activating mutations of Notch1 (N1) ...

Pulmonary surfactant protein A (SP‐A) is an oligomeric collectin that recognizes lipid and carbohydrate moieties present on broad range of micro‐organisms, and mediates microbial lysis and clearance. SP‐A also modulates multiple immune‐related functions including cytokine production and chemotaxis for phagocytes. Here we describe the molecular interaction between the extracellular matrix protein microfibril‐associated protein 4 (MFAP4) and SP‐A. MFAP4 is a collagen‐binding molecule containing a C‐terminal fibrinogen‐like domain and a N‐terminal located integrin‐binding motif. We produced recombinant MFAP4 with a molecular mass of 36 and 66 kDa in the reduced and unreduced states respectively. Gel filtration chromatography and chemical crosslinking showed that MFAP4 forms oligomers of four dimers. We demonstrated calcium‐dependent binding between MFAP4 and human SP‐A1 and SP‐A2. No binding was seen to recombinant SP‐A composed of the neck region and carbohydrate recognition domain of SP‐A indicating that the interaction between MFAP4 and SP‐A is mediated via the collagen domain of SP‐A. Monoclonal antibodies directed against MFAP4 and SP‐A were used for immunohistochemical analysis, which demonstrates that the two molecules colocalize both on the elastic fibres in the interalveolar septum and in elastic lamina of pulmonary arteries of chronically inflamed lung tissue. We conclude, that MFAP4 interacts with SP‐A via the collagen region in vitro, and that MFAP4 and SP‐A colocates in different lung compartments indicating that the interaction may be operative in vivo.

Elastic fibers are composed of the protein elastin and a network of 10 -12-nm microfibrils, which are composed of several glycoproteins, including fibrillin-1, fibrillin-2, and MAGP1/2 (microfibril-associated glycoproteins-1 and -2). Although fibrillins and MAGPs covalently associate, we find that the DSL (Delta/Serrate/LAG2) protein Jagged1, an activating ligand for Notch receptor signaling, also interacts with MAGP-2 in both yeast two-hybrid and coimmunoprecipitation studies. Interaction between Jagged1 and MAGP-2 requires the epidermal growth factor-like repeats of Jagged1. MAGP-2 was found complexed with the Jagged1 extracellular domain shed from 293T cells and COS-7 cells coexpressing fulllength Jagged1 and MAGP-2. MAGP-2 shedding of the Jagged1 extracellular domain was decreased by the metalloproteinase hydroxamate inhibitor BB3103 implicating proteolysis in its release. Although MAGP-2 also interacted with the other DSL ligands, Jagged2 and Delta1, they were not found associated with MAGP-2 in the conditioned media, identifying differential effects of MAGP-2 on DSL ligand shedding. The related microfibrillar protein MAGP-1 was also found to interact with DSL ligands but, unlike MAGP-2, was unable to facilitate the shedding of Jagged1. Our findings suggest that in addition to its role in microfibrils, MAGP-2 may also affect cellular differentiation through modulating the Notch signaling pathway either by binding to cell surface DSL ligands or by facilitating release and/or stabilization of a soluble extracellular form of Jagged1.

We found that E-cadherin and epidermal growth factor receptor (EGFR) are associated in mammary epithelial cells and that E-cadherin engagement in these cells induces transient activation of EGFR, as previously seen in keratinocytes (37). In contrast, EGFR does not associate with and is not activated by N-cadherin. Analysis of cells expressing chimeric cadherins revealed that the extracellular domain of E-cadherin is required for interaction with and activation of EGFR. This activation results in tyrosine phosphorylation of known EGFR substrates and reduction in focal adhesions. These interactions, however, are not necessary for suppression of cell motility by E-cadherin.