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

Abstract

Background: 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 NEDD4L that generates isoforms that contain or lack a C2 domain.

Results: To address the potential functional significance of the NEDD4L 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 Xenopus laevis 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 beta-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.

Conclusion: 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.

Figure 1

NEDD4L is expressed as isoforms that contain (NEDD4L-C2(+)) or lack (NEDD4L-C2(-)) a calcium binding C2 domain. (A) Chromosome 18q21.31 where NEDD4L transcription occurs from several major alternate promoters designated p1-4 and generates NEDD4L-C2(+) and NEDD4L-C2(-) isoforms. Several additional promoters that generate NEDD4L-C2(+) and NEDD4L-C2(-) isoforms exist [11]. The location of rs4149601 is labeled on the p2 transcript. (B) The C2(+) domain transcript that arises from p1 encodes a methionine in exon 1, initiating translation upstream of the C2 domain thereby generating NEDD4L-C2(+) isoforms. The p2 transcript generates NEDD4L-C2(+) and NEDD4L-C2(-) isoforms depending on the identity of the rs4149601 variant [11]. Whereas the G variant generates NEDD4L-C2(+) and NEDD4L-C2(-), the A variant only generates NEDD4L-C2(-) [11]. Transcripts from p3 and p4 encode only NEDD4L-C2(-) utilizing an initiation codon in exon 7, downstream of the C2 domain. (C) The domain architecture of human NEDD4L-C2(+) and NEDD4L-C2(-). Each isoform contains four WW domains and a HECT (homologous to E6 associated protein carboxy terminus) domain.

Figure 2

Differential subcellular localization of EGFP tagged NEDD4L-C2(+) and NEDD4L-C2(-) isoforms. Confocal microscopic images of X. laevis A6 cells transiently transfected with EGFP-NEDD4L-C2(+) (A), NEDD4L-C2(+)-EGFP (B), EGFP (C), EGFP-NEDD4L-C2(-) (D), and NEDD4L-C2(-)-EGFP (E). Western blot experiments of crude lysates from transiently transfected A6 cells (F) demonstrate that differential degradation of either isoform did not occur. The monoclonal EGFP antibody, JL-8 (Clontech) was used for detection. Confocal microscopic images of A6 cells transiently transfected with EGFP-NEDD4L-C2(-) and incubated with the early endosomal marker, Transferrin-Texas Red^® (G-I) indicate that EGFP-NEDD4L-C2(-) localizes to the early endosome. Green (EGFP-tagged NEDD4L-C2(-)) and blue (nuclear marker) channels (G). Red (early endosome marker) and blue channels (H). Green, blue and red channel overlay (I). Scale bars are equivalent to 10 μm.

Figure 3

Subcellular localization of EGFP tagged NEDD4L-C2(+) and NEDD4L-C2(-) isoforms in response to ionomycin and Ca²⁺ treatment. Confocal microscopic images of live A6 cells transiently transfected with EGFP (A, B), EGFP-PKCα-C2 (C, D), EGFP-NEDD4L-C2(-) (E, F), and EGFP-NEDD4L-C2(+) (G, H). Images prior to (A, C, E & G) and 90 seconds after (B, D, F, H) incubation with10 μM ionomycin and 1.97 mM Ca²⁺. EGFP and EGFP-NEDD4L-C2(-) do not relocalize in response to an intracellular Ca²⁺ influx. EGFP-PKCα-C2 relocalizes to the plasma membrane. EGFP-NEDD4L-C2(+) mobilizes to numerous small intracellular structures. Scale bars are equivalent to 10 μm.

Figure 4

Subcellular localization of the EGFP tagged NEDD4L-C2(+) isoform and the NEDD4L C2 domain (C2(only)) in response to ionomycin and Ca²⁺ treatment. Confocal images of live A6 cells transiently transfected with EGFP-NEDD4L-C2(+) (A-C) or EGFP-C2(only) (D-F) that were incubated in 1.97 Ca²⁺. Images were acquired at 0 (A), 40 (B), 90 (C), 0 (D), 10 (E), and 30 (F) seconds after a 10 uM ionomycin addition. Both EGFP-NEDD4L-C2(+) and EGFP-C2(only) respond to the Ca²⁺ stimulus by first mobilizing to the plasma membrane and then relocalizing to numerous small intracellular structures. Scale bars are equivalent to 10 μm.

Figure 5

Distinct subcellular localization behaviors of EGFP tagged α- and β-ENaC and mCherry tagged NEDD4L-C2(+) or NEDD4L-C2(+)-C943A in response to an ionomycin treatment in the presence of Ca²⁺. Confocal images of live A6 cells stably expressing α-ENaC-EGFP (A-E) or EGFP-β-ENaC (F-J, K-O) transiently transfected with mCherry-NEDD4L-C2(+) (A-E, F-J) or mCherry-NEDD4L-C2(+)-C943A (K-O) and incubated in 1.97 mM Ca²⁺. The images were acquired at 0 (A), 30 (B), 210 (C), 420 (D, E), 0 (F), 41 (G), 66 (H), 236 (I, J), 0 (K), 80 (L), 120 (M), and 240 (N, O)seconds after a 10 μM ionomycin addition. All images are overlays of the blue, green and red channels except for images E, J, and Owhich show only the blue and green channels.

Figure 6

Summary schematic of the observed mobilization response of NEDD4L-C2(+) in response to a Ca²⁺ stimulus. NEDD4L-C2(+)-C943A that lacks ubiquitin ligase activity does not mobilize to the plasma membrane in step 4, and prevents β-ENaC transit to the plasma membrane in step 4.