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. 2014 Feb 18;111(7):2578-83.
doi: 10.1073/pnas.1319947111. Epub 2014 Feb 3.

BMP Signaling Requires Retromer-Dependent Recycling of the Type I Receptor

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Free PMC article

BMP Signaling Requires Retromer-Dependent Recycling of the Type I Receptor

Ryan J Gleason et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The transforming growth factor β (TGFβ) superfamily of signaling pathways, including the bone morphogenetic protein (BMP) subfamily of ligands and receptors, controls a myriad of developmental processes across metazoan biology. Transport of the receptors from the plasma membrane to endosomes has been proposed to promote TGFβ signal transduction and shape BMP-signaling gradients throughout development. However, how postendocytic trafficking of BMP receptors contributes to the regulation of signal transduction has remained enigmatic. Here we report that the intracellular domain of Caenorhabditis elegans BMP type I receptor SMA-6 (small-6) binds to the retromer complex, and in retromer mutants, SMA-6 is degraded because of its missorting to lysosomes. Surprisingly, we find that the type II BMP receptor, DAF-4 (dauer formation-defective-4), is retromer-independent and recycles via a distinct pathway mediated by ARF-6 (ADP-ribosylation factor-6). Importantly, we find that loss of retromer blocks BMP signaling in multiple tissues. Taken together, our results indicate a mechanism that separates the type I and type II receptors during receptor recycling, potentially terminating signaling while preserving both receptors for further rounds of activation.

Keywords: C. elegans; endocytosis; receptor trafficking.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
AP-2 adaptor complex mutants, dpy-23(e480) and apa-2(ox422), display reduced body size phenotypes, inhibit Sma/Mab signaling, and block receptor internalization of SMA-6::GFP. (A) Schematic depiction of the C. elegans intestine to demonstrate focal planes captured to study SMA-6 and DAF-4 localization. White arrowheads indicate lateral membrane, and yellow arrowheads indicate apical lumen of the intestine. (BD) Micrographs of SMA-6::GFP expressed in the intestine to compare localization in control L4440(RNAi), apa-2(RNAi), and dpy-23(RNAi). On the top (basolateral) focal plane, arrowheads indicate lateral membrane. (E) Quantification of SMA-6::GFP micrographs (n = 6). (F) Body length of N2 wild-type, sma-6(wk7), dpy-23(e480), apa-2(ox422), and transgenic rescue strain pelt-3::SMA-6::GFP; sma-6(wk7). (G) Expression of the RAD-SMAD GFP reporter in wild-type, sma-6(wk7), dpy-23(e480), and apa-2(ox422). Staged at larval stage L3. (n = 6). (H) qRT-PCR of intestinally expressed genes F35C5.9 and R09H10.5 in wild-type, sma-6(wk7), dpy-23(e480), and apa-2(ox422). (IK) Micrographs of DAF-4::GFP expressed in the intestine to compare localization in control L4440 RNAi, apa-2(RNAi), and dpy-23(RNAi). On the top (basolateral) focal plane, arrowheads indicate lateral membrane. (L) Quantification of DAF-4::GFP micrographs (n = 6). Error bars, SEM. ***P < 0.001. See also Fig. S1.
Fig. 2.
Fig. 2.
Disparate phenotypes of DAF-4::GFP and SMA-6::GFP in the absence of endocytic recycling protein RME-1, retromer complex mutants vps-35(hu68) and snx-3(tm1595), and recycling endosome mutant arf-6(tm1447). (AG) Micrographs of SMA-6::GFP expressed in the intestine to compare localization in wild-type, rme-1(b1045), vps-35(hu68), snx-3(tm1595), snx-1(tm847), snx-27(tm5356), and arf-6(tm1447). On the top (basolateral) focal plane, white arrowheads indicate lateral membrane. (H) Quantification of SMA-6::GFP micrographs (n = 6). (I) Expression of the RAD-SMAD GFP reporter in wild-type, sma-6(wk7), vps-35(hu68), and rme-1(b1045) staged at L3 (n = 6). (J) qRT-PCR of intestinally expressed genes F35C5.9 and R09H10.5 in wild-type, sma-6(wk7), rme-1(b1045), and vps-35(hu68). (K) Body length of N2 wild-type, sma-6(wk7), rme-1(b1045), vps-35(hu68), and arf-6(tm1447). (LO) Micrographs of DAF-4::GFP expressed in the intestine to compare localization in wild-type, rme-1(b1045), arf-6(tm1447), and vps-35(hu68) in the middle (midsagittal cross-section) focal plane. Yellow arrowheads indicate apical lumen of the intestine. (L′–O′) Magnified regions annotated by dotted squares in LO. Arrows indicate aberrant accumulation in mutant backgrounds. (P) Quantification of DAF-4::GFP micrographs (n = 6). (Q) Expression of the RAD-SMAD GFP reporter in wild-type, sma-6(wk7), and arf-6(tm1447) staged at L3 (n = 6). Error bars, SEM. ***P < 0.001; *P ≤ 0.05. See also Fig. S2.
Fig. 3.
Fig. 3.
SMA-6 is mislocalized to the lysosome when retromer-dependent recycling is impaired. (A-A″) Colocalization of SMA-6::GFP with TagRFP::RAB-7 expressed in the intestine to compare localization in wild-type in the middle (midsagittal cross-section) focal plane. Yellow arrowheads indicate apical lumen of the intestine. (A″′) Magnified image of A″ is designated by dashed rectangular outline. (BB″) Colocalization of SMA-6::GFP with TagRFP::RAB-7 in vps-35(hu68) in the middle (midsagittal cross-section) focal plane. Yellow arrowheads indicate apical lumen of the intestine. (B″′) Magnified image of B″ designated by dashed rectangular outline. (C) Quantification of SMA-6::GFP colocalization with TagRFP::RAB-7. (D) Pearson and Mander’s coefficients for colocalization of SMA-6::GFP with TagRFP::RAB-7. n = 6. Error bars, SEM. ***P < 0.001.
Fig. 4.
Fig. 4.
Retromer-dependent recycling occurs after biosynthesis and internalization. (A and B) Micrographs of SMA-6::GFP to compare localization on the top (basolateral) focal plane in control L4440(RNAi), dpy-23(RNAi). White arrowheads indicate lateral membrane. (C) Quantification of SMA-6::GFP micrographs from A and B (n = 6). (D and E) Micrographs of vps-35(hu68);SMA-6::GFP to compare localization on the top (basolateral) focal plane in control L4440(RNAi), dpy-23(RNAi). White arrowheads indicate lateral membrane. (F) Quantification of vps-35(hu68);SMA-6::GFP micrographs from D and E (n = 6). (G and H) Micrographs of SMA-6::GFP to compare localization in control L4440(RNAi), cup-5(RNAi) in the middle (midsagittal cross-section) focal plane. Yellow arrowheads indicate apical lumen of the intestine. (I) Quantification of SMA-6::GFP micrographs from G and H (n = 6). (J and K) Micrographs of vps-35(hu68); SMA-6::GFP to compare localization in control L4440(RNAi), cup-5(RNAi) in the middle (midsagittal cross-section) focal plane. Yellow arrowheads indicate apical lumen of the intestine. (L) Quantification of vps-35(hu68); SMA-6::GFP micrographs from J and K (n = 6). Error bars, SEM. ***P < 0.001; **P ≤ 0.01.
Fig. 5.
Fig. 5.
The retromer complex binds the intracellular domain of SMA-6. (A) Glutathione beads loaded with recombinant GST or GST-SMA-6 intracellular domain were incubated with a lysate prepared from transgenic worms expressing GFP::VPS-35. Unbound proteins were washed away, and bound proteins were eluted with Laemmli sample buffer, separated by SDS/PAGE, and analyzed by Western blot with anti-GFP antibody. The GFP::VPS-35 band observed in worms at 120 kDa was bound by the GST-SMA-6 intracellular domain, but not by GST alone. Input lanes contain 10% (vol/vol) worm lysate used in the binding assays. Loading of bait GST (26 kDa) or GST-SMA-6 (100 kDa) was visualized by Ponceau S. (B) Purified recombinant FLAG(FLAG epitope tag)-tagged retromer complex [consisting of the proteins (3xFLAG)Vps26-(3xFLAG)Vps29-(3xFLAG)Vps35-His6] incubated with purified GST or GST fusion proteins bearing the wild-type intracellular domains of SMA-6 and CI-MPR as control. Proteins were pulled down with glutathione-Sepharose beads, bound FLAG-tagged retromer components were detected with an antibody to the FLAG-tag, and proteins were visualized with Ponceau S.

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