A human polymorphism affects NEDD4L subcellular targeting by leading to two isoforms that contain or lack a C2 domain

<p>Abstract</p> <p>Background</p> <p>Ubiquitination serves multiple cellular functions, including proteasomal degradation and the control of stability, function, and intracellular localization of a wide variety of proteins. NEDD4L is a member of the HECT class of E3 ubiquitin ligases. A defining feature of NEDD4L protein isoforms is the presence or absence of an amino-terminal C2 domain, a class of subcellular, calcium-dependent targeting domains. We previously identified a common variant in human <it>NEDD4L </it>that generates isoforms that contain or lack a C2 domain.</p> <p>Results</p> <p>To address the potential functional significance of the <it>NEDD4L </it>common variant on NEDD4L subcellular localization, NEDD4L isoforms that either contained or lacked a C2 domain were tagged with enhanced green fluorescent protein, transfected into <it>Xenopus laevis </it>kidney epithelial cells, and imaged by performing confocal microscopy on live cells. We report that the presence or absence of this C2 domain exerts differential effects on the subcellular distribution of NEDD4L, the ability of C2 containing and lacking NEDD4L isoforms to mobilize in response to a calcium stimulus, and the intracellular transport of subunits of the NEDD4L substrate, ENaC. Furthermore, the ability of the C2-containing isoform to influence β-ENaC mobilization from intracellular pools involves the NEDD4L active site for ubiquitination. We propose a model to account for the potential impact of this common genetic variant on protein function at the cellular level.</p> <p>Conclusion</p> <p>NEDD4L isoforms that contain or lack a C2 domain target different intracellular locations. Additionally, whereas the C2-containing NEDD4L isoform is capable of shuttling between the plasma membrane and intracellular compartments in response to calcium stimulus the C2-lacking isoform can not. The C2-containing isoform differentially affects the mobilization of ENaC subunits from intracellular pools and this trafficking step requires NEDD4L ubiquitin ligase activity. This observation suggests a new mechanism for the requirement for the PY motif in cAMP-mediated exocytosis of ENaC. We have elucidated how a common genetic variant can underlie significant functional diversity in NEDD4L at the cellular level. We propose a model that describes how that functional variation may influence blood pressure. Moreover, our observations regarding differential function of the NEDD4L isoforms may impact other aspects of physiology that involve this ubiquitin ligase.</p

Collagènes et laminines : quels messages pour quelles cellules ?

International audienceCells in animal tissues are in contact with a structured set of well-defined proteins that constitute the extracellular matrix. Among these proteins, collagens are ubiquitous in distribution, whereas laminins are found only in basement membranes. In addition to their structural role, collagens and laminins convey messages to cells. A discussion is presented of the structure and diversity of collagens (about 20 types) and laminins (about 12 types). The nature of the cell receptors involved in message delivery (integrins and non-integrins) and the mechanisms possibly responsible for signal transduction are reviewed

ULTRASTRUCTURE OF FILAMENTS OF SPONGE IRCINIA-VARIABILIS OS

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Fibronectin-like protein in Porifera: its role in cell aggregation.

Experiments were carried out on a freshwater sponge (Ephydatia mulleri) in order to demonstrate the presence of fibronectin in Porifera. By using antibodies to highly purified human plasma fibronectin, the presence of a similar or identical protein could be demonstrated in the membranes of E. mulleri cells such as epithelial cells, fibroblast-like cells, and choanocytes. The reaction was specific, could be abolished by the addition of excess fibronectin, and was not observed with nonimmune rabbit serum. The immune fluorescent reaction became stronger when the sponge cells were pretreated with acetone and could also be observed, although with a less intense staining, on the intercellular matrix. This shows the predominant presence of a sponge fibronectin-like protein in the cell membranes and also its presence to a lesser extent in the intercellular matrix. When dissociated sponge cells were led to reassociate under the microscope, reassociation could be completely inhibited by anti-human fibronectin antiserum up to a dilution of 1:120 and partially inhibited up to a dilution of 1:240. The reassociation of dissociated sponge cells could also be inhibited by the addition of purified gelatin but not with serum albumin or with a normal, nonimmune rabbit serum. These results clearly indicate that a sponge cell fibronectin-like protein may play an important role as the (or one of the) recognition site(s) of the aggregation factor(s) and can therefore be directly involved in cell association, morphogenesis, and differentiation