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Proteins containing a membrane-anchored metalloprotease domain, a disintegrin domain, and a cysteine-rich region (MDC proteins) are thought to play an important role in mammalian fertilization, as well as in somatic cell-cell interactions. We have identified PCR sequence tags encoding the disintegrin domain of five distinct MDC proteins from Xenopus laevis testis cDNA. Four of these sequence tags (xMDC9, xMDC11.1, xMDC11.2, and xMDC13) showed strong similarity to known mammalian MDC proteins, whereas the fifth (xMDC16) apparently represents a novel family member. Northern blot analysis revealed that the mRNA for xMDC16 was only expressed in testis, and not in heart, muscle, liver, ovaries, or eggs, whereas the mRNAs corresponding to the four other PCR products were expressed in testis and in some or all somatic tissues tested. The xMDC16 protein sequence, as predicted from the full-length cDNA, contains a metalloprotease domain with the active-site sequence HEXXH, a disintegrin domain, a cysteine-rich region, an EGF repeat, a transmembrane domain, and a short cytoplasmic tail. To study a potential role for these xMDC proteins in fertilization, peptides corresponding to the predicted integrin-binding domain of each protein were tested for their ability to inhibit X. laevis fertilization. Cyclic and linear xMDC16 peptides inhibited fertilization in a concentration-dependent manner, whereas xMDC16 peptides that were scrambled or had certain amino acid replacements in the predicted integrin-binding domain did not affect fertilization. Cyclic and linear xMDC9 peptides and linear xMDC13 peptides also inhibited fertilization similarly to xMDC16 peptides, whereas peptides corresponding to the predicted integrin-binding site of xMDC11.1 and xMDC11.2 did not. These results are discussed in the context of a model in which multiple MDC protein-receptor interactions are necessary for fertilization to occur.

A disintegrin and metalloproteinase 17 (ADAM17) controls the release of the pro-inflammatory cytokine tumor necrosis factor α (TNFα, also known as TNF) and is crucial for protecting the skin and intestinal barrier by proteolytic activation of epidermal growth factor receptor (EGFR) ligands. The seven-membrane-spanning protein called inactive rhomboid 2 (Rhbdf2; also known as iRhom2) is required for ADAM17-dependent TNFα shedding and crosstalk with the EGFR, and a point mutation (known as sinecure, sin) in the first transmembrane domain (TMD) of Rhbdf2 (Rhbdf2sin) blocks TNFα shedding, yet little is known about the underlying mechanism. Here, we used a structure-function analysis informed by structural modeling to evaluate the interaction between the TMD of ADAM17 and the first TMD of Rhbdf2, and the role of this interaction in Rhbdf2-ADAM17-dependent shedding. Moreover, we show that double mutant mice that are homozygous for Rhbdf2sin/sin and lack Rhbdf1 closely resemble Rhbdf1/2-/-double knockout mice, highlighting the severe functional impact of the Rhbdf2sin/sin mutation on ADAM17 during mouse development. Taken together, these findings provide new mechanistic and conceptual insights into the critical role of the TMDs of ADAM17 and Rhbdf2 in the regulation of the ADAM17 and EGFR, and ADAM17 and TNFα signaling pathways. [ABSTRACT FROM AUTHOR]

The metalloproteinase and major amyloid precursor protein (APP) α-secretase candidate ADAM10 is responsible for the shedding of proteins important for brain development, such as cadherins, ephrins, and Notch receptors. Adam10-/- mice die at embryonic day 9.5, due to major defects in development of somites and vasculogenesis. To investigate the function of ADAM10 in brain, we generated Adam10 conditional knock-out (cKO) mice using a Nestin-Cre promotor, limiting ADAM10 inactivation to neural progenitor cells (NPCs) and NPC-derived neurons and glial cells. The cKO mice die perinatally with a disrupted neocortex and a severely reduced ganglionic eminence, due to precocious neuronal differentiation resulting in an early depletion of progenitor cells. Premature neuronal differentiation is associated with aberrant neuronal migration and a disorganized laminar architecture in the neocortex. Neurospheres derived from Adam10 cKO mice have a disrupted sphere organization and segregated more neurons at the expense of astrocytes. We found that Notch-1 processing was affected, leading to downregulation of several Notch-regulated genes in Adam10 cKO brains, in accordance with the central role of ADAM10 in this signaling pathway and explaining the neurogenic phenotype. Finally, we found that α-secretase-mediated processing of APP was largely reduced in these neurons, demonstrating that ADAM10 represents the most important APP α-secretase in brain. Our study reveals that ADAM10 plays a central role in the developing brain by controlling mainly Notch-dependent pathways but likely also by reducing surface shedding of other neuronal membrane proteins including APP. [ABSTRACT FROM AUTHOR]

Protein ectodomain shedding, the proteolytic release of the extracellullar domain of membrane-tethered proteins, can dramatically affect the function of cell surface receptors, growth factors, cytokines, and other proteins. In this study, we evaluated the activities involved in ectodomain shedding of p75NTR, a neurotrophin receptor with critical roles in neuronal differentiation and survival, p75NTR is shed in a variety of cell types, including dorsal root ganglia cells and PC12 cells. In Chinese hamster ovary cells, inhibitors of the MEK/ERK and p38 MAP kinase pathways uncovered distinct signaling pathways required for the constitutive and stimulated shedding of p75NTR. Stimulated p75NTR shedding is abrogated in M2 mutant Chinese hamster ovary cells that lack functional tumor necrosis factor-α converting enzyme (TACE, also referred to as ADAM17) and in cells isolated from adam17-/- mice, but not in cells from adam9/12/15-/- or adam10-/- mice. Stimulated p75NTR shedding is strongly reduced by deletion of 15 amino acid residues in its extracellular membrane-proximal stalk domain. However, similar to other shed proteins, point mutations and overlapping shorter deletions within this region have little or no effect on shedding. Because ectodomain shedding of p75NTR releases a soluble ectodomain and could also be a prerequisite for its regulated intramembrane proteolysis, these findings may have important implications for the functional regulation of p75NTR. [ABSTRACT FROM AUTHOR]

Congenital heart disease is the most common form of human birth defects, yet much remains to be learned about its underlying causes. Here we report that mice lacking functional ADAM19 (mnemonic for a disintegrin and metalloprotease 19) exhibit severe defects in cardiac morphogenesis, including a ventricular septal defect (VSD), abnormal formation of the aortic and pulmonic valves, leading to valvular stenosis, and abnormalities of the cardiac vasculature. During mouse development, ADAM19 is highly expressed in the conotruncus and the endocardial cushion, structures that give rise to the affected heart valves and the membranous ventricular septum. ADAM19 is also highly expressed in osteoblast-like cells in the bone, yet it does not appear to be essential for bone growth and skeletal development. Most adam19[sup -/-] animals die perinatally, likely as a result of their cardiac defects. These findings raise the possibility that mutations in ADAM19 may contribute to human congenital heart valve and septal defects. [ABSTRACT FROM AUTHOR]

A disintegrin and metalloproteinase 17 (ADAM17) controls the release of the pro-inflammatory cytokine tumor necrosis factor α (TNFα, also known as TNF) and is crucial for protecting the skin and intestinal barrier by proteolytic activation of epidermal growth factor receptor (EGFR) ligands. The seven-membrane-spanning protein called inactive rhomboid 2 (Rhbdf2; also known as iRhom2) is required for ADAM17-dependent TNFα shedding and crosstalk with the EGFR, and a point mutation (known as sinecure, sin) in the first transmembrane domain (TMD) of Rhbdf2 (Rhbdf2 sin ) blocks TNFα shedding, yet little is known about the underlying mechanism. Here, we used a structure-function analysis informed by structural modeling to evaluate the interaction between the TMD of ADAM17 and the first TMD of Rhbdf2, and the role of this interaction in Rhbdf2-ADAM17-dependent shedding. Moreover, we show that double mutant mice that are homozygous for Rhbdf2 sin/sin and lack Rhbdf1 closely resemble Rhbdf1/2 -/- double knockout mice, highlighting the severe functional impact of the Rhbdf2 sin/sin mutation on ADAM17 during mouse development. Taken together, these findings provide new mechanistic and conceptual insights into the critical role of the TMDs of ADAM17 and Rhbdf2 in the regulation of the ADAM17 and EGFR, and ADAM17 and TNFα signaling pathways., (© 2017. Published by The Company of Biologists Ltd.)

All ligands of the epidermal growth factor receptor (EGFR) are made as membrane anchored precursors that can be proteolytically processed and released from the plasma membrane. This process, which is referred to as protein ectodomain shedding, is emerging as a key regulator of the function of EGFR ligands. In light of the important roles of EGFR signaling in development and disease, it will be important to understand more about the regulation of proteolytic processing of EGFR ligands. This chapter describes a sensitive and semiquantitative method to measure ectodomain shedding of EGFR ligands that was designed to facilitate studies of this process in cells.

Protein ectodomain shedding, the proteolytic release of the extracellullar domain of membrane-tethered proteins, can dramatically affect the function of cell surface receptors, growth factors, cytokines, and other proteins. In this study, we evaluated the activities involved in ectodomain shedding of p75NTR, a neurotrophin receptor with critical roles in neuronal differentiation and survival. p75NTR is shed in a variety of cell types, including dorsal root ganglia cells and PC12 cells. In Chinese hamster ovary cells, inhibitors of the MEK/ERK and p38 MAP kinase pathways uncovered distinct signaling pathways required for the constitutive and stimulated shedding of p75NTR. Stimulated p75NTR shedding is abrogated in M2 mutant Chinese hamster ovary cells that lack functional tumor necrosis factor-alpha converting enzyme (TACE, also referred to as ADAM17) and in cells isolated from adam17-/- mice, but not in cells from adam9/12/15-/- or adam10-/- mice. Stimulated p75(NTR) shedding is strongly reduced by deletion of 15 amino acid residues in its extracellular membrane-proximal stalk domain. However, similar to other shed proteins, point mutations and overlapping shorter deletions within this region have little or no effect on shedding. Because ectodomain shedding of p75NTR releases a soluble ectodomain and could also be a prerequisite for its regulated intramembrane proteolysis, these findings may have important implications for the functional regulation of p75NTR.