BMP signaling requires retromer-dependent recycling of the type I receptor

Significance

The mechanisms that mediate bone morphogenetic protein (BMP) receptor recycling, and the importance of such recycling for signaling in vivo, have remained poorly understood. We find that the retromer complex functions as a linchpin in the recycling of the BMP type I receptor SMA-6 (small-6). In the absence of retromer-dependent recycling, retromer mutants result in the missorting of SMA-6 to lysosomes and a loss of BMP-mediated signaling. Surprisingly, we find that the BMP type II receptor, DAF-4 (dauer formation-defective-4), recycles through a distinct recycling pathway. 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.

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.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β (TGFβ) superfamily of ligands that regulate an array of early developmental processes across metazoan phylogenies. Aberrant BMP signaling results in tumorigenesis in multiple tissues and also contributes to a variety of other important disorders (1). BMP ligands signal through a heteromeric complex of two transmembrane serine–threonine kinase receptors, referred to as the type I and type II receptors. On binding of the ligand to the receptors, a series of signaling events culminate in regulating gene expression.

The output of conserved signal transduction pathways, including those mediated by epidermal growth factor receptor, Notch, and G protein-coupled receptors, depend not only on the activation of these receptors by extracellular stimuli but also on the endocytic internalization and postendocytic trafficking of the receptors, which regulates the availability and compartmentalization of the signal transduction machinery (2–4). Once endocytosed into early endosomes, signal transduction receptors are either sorted into a recycling pathway that will return the molecule to the cell surface for another round of signaling or are sorted into a degradative pathway via multivesicular bodies and late endosomes to be degraded in the lysosome. Although initial studies to identify the molecular complexes that regulate TGFβ receptor recycling have focused on the type II receptor and are limited, reports have shown that recycling of the type II receptor is mediated by recycling endosomes (5, 6).

In Caenorhabditis elegans, a conserved BMP signaling pathway, the Sma/Mab pathway, regulates diverse developmental processes including cell/body size, male-tail morphogenesis, dorsoventral cell patterning, immune regulation, and olfactory learning, among others (7–10). In the C. elegans Sma/Mab pathway, the secreted ligand DBL-1 (decapentaplegic/bone morphogenetic protein-like-1) binds the type II, DAF-4 (dauer formation-defective-4), and type I, SMA-6 (small-6), receptor complex, and DAF-4 phosphorylates SMA-6, which in turn phosphorylates key residues on SMAD (small and mothers against decapentaplegic) proteins, allowing them to accumulate in the nucleus and activate or repress target gene transcription. The DBL-1 signal is received by SMA-6/DAF-4 complexes expressed in the hypodermis, intestine, and other peripheral tissues.

Some studies of TGFβ trafficking and signaling in mammalian Mv1Lu cells have indicated that TGFβ signaling requires clathrin-mediated internalization of activated receptors to transduce signals to the nucleus via SMADs, presumably because receptor–SMAD interaction requires early endosome adapters (11). However, other studies in the same cell line report the opposite, that blocking clathrin-dependent endocytosis of TGFβ-receptors enhances signal transduction (12). Thus, it remained important to test the requirements for receptor endocytosis in transducing TGFβ signals in an intact animal model such as C. elegans. We also set out both to identify molecular sorting complexes that regulate BMP receptor type I and II recycling and to determine how receptor recycling affects signaling. Our in vivo results provide strong evidence that clathrin-dependent endocytosis is necessary for BMP signaling in C. elegans. Furthermore, we find that after internalization, two distinct recycling pathways regulate the transport of the type I and type II receptors back to the cell surface. Recycling of the type I receptor is regulated by the retromer complex, whereas the type II receptor is recycled via a distinct recycling pathway regulated by ARF-6 (ADP-ribosylation factor-6). In addition, we found that the type I receptor cytoplasmic tail binds directly to the retromer complex. Our work establishes a direct link between retromer-dependent recycling and BMP signaling in vivo, identifies distinct recycling pathways for the type I and type II receptors, and provides a genetically tractable system to study the regulation of vesicle trafficking on the BMP signaling pathway.

Results

Clathrin-Dependent Endocytosis Is Necessary for BMP Receptor Internalization and Signaling.

To test the requirements for receptor internalization on signal transduction within intact animals in vivo, we determined the effects of loss of clathrin-adapter protein (AP)-2 subunits on Sma/Mab pathway signaling in C. elegans. We found that mutants lacking C. elegans μ2-adaptin (DPY-23) or α2-adaptin (APA-2) displayed body size defects as severe as those in animals completely lacking the type I receptor SMA-6 (Fig. 1F). Furthermore, molecular analysis confirmed this interpretation, indicating a severe block in Sma/Mab signaling in the hypodermis and intestine of dpy-23 and apa-2 mutants. This included analysis of a hypodermal expression of a concatamer of smad-binding elements driving GFP [the reporter acting downstream of SMAD (RAD-SMAD) reporter] and quantitative RT-PCR (qRT-PCR) analysis of transcript levels of two intestine-specific genes whose expression levels are regulated by the Sma/Mab pathway (Fig. 1 G and H) (13–15).

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. (B–D) 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). (I–K) 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.

If these effects are mediated through the receptors, we would expect to find BMP receptors trapped at the cell surface under these conditions. We determined the subcellular localization of SMA-6 and DAF-4 in the large, well-characterized epithelial cells of the C. elegans intestine, using low-copy number transgenes driven by an intestine-specific promoter (Fig. 1A). GFP-tagged SMA-6 and DAF-4 are functional, as shown in this and previous work (Fig. 1F) (16). We found that both SMA-6::GFP and DAF-4::GFP, visualized in otherwise wild-type intact living animals, localized to the basolateral plasma membrane, where they are in position to receive signaling molecules secreted by neurons (Fig. 1 B and I). SMA-6::GFP and DAF-4::GFP also labeled intracellular puncta, at least some of which we identified as endosomes.

We determined that SMA-6::GFP accumulated to much higher levels on the intestinal basolateral plasma membrane in animals depleted of AP-2 subunits by RNAi, indicating that SMA-6 requires AP-2 for endocytosis (Fig. 1 B–E). However, DAF-4 surface levels did not change in response to depletion of AP-2, suggesting that DAF-4 is AP-2-independent (Fig. 1 I–L). Previous studies of BMP receptor internalization in mammalian cell culture indicated that the type II receptor was internalized via clathrin-dependent and clathrin-independent mechanisms, whereas the type I receptor was strictly clathrin-dependent (11, 17, 18). Thus, type II receptor DAF-4 may be internalized by clathrin-independent mechanisms or may use alternative clathrin adapters. Further analysis demonstrated that surface levels of SMA-6 and DAF-4 did not increase in animals devoid of the ligand DBL-1, suggesting that receptor internalization does not require ligand binding (Fig. S1). We conclude that AP-2-dependent endocytosis of the type I receptor SMA-6 is necessary for signal transduction in the Sma/Mab pathway.

Postendocytic Trafficking and Signaling of the BMP Type I and Type II Receptors Are Regulated by Distinct Recycling Pathways.

Once internalized by endocytosis, receptors are trafficked to early endosomes, from which they may be recycled to the plasma membrane or delivered to the lysosome. Several recycling pathways exist, including routes through the endocytic recycling compartment (ERC) and/or the trans-Golgi network (19). RME-1 is a founding member of the conserved EHD/RME-1 (Eps15 homology-domain containing/receptor-mediated endocytosis-1) protein family and is required for a variety of recycling events, including ERC to plasma membrane transport and endosome to Golgi transport (20, 21). Importantly, we found that loss of RME-1 resulted in dramatically different defects in the subcellular localization of SMA-6 and DAF-4; DAF-4::GFP accumulated in intracellular vesicles, whereas overall levels of SMA-6::GFP were severely reduced, suggesting that SMA-6 was being inappropriately degraded (Fig. 2 A, B, L, and M). Previous work indicated that a block in recycling to the plasma membrane via the ERC often results in intracellular trapping of receptors, whereas blocks in retromer-dependent recycling often results in missorting of receptors to the lysosome, where they are degraded (20, 22–24). Consistent with this idea, the accumulation of intracellular DAF-4 in the intestine of rme-1 mutants strongly resembled the accumulation of well-characterized ERC cargo hTAC::GFP (human IL-2 receptor α-chain) in rme-1 mutants (Fig. S2 A–C). The loss of SMA-6::GFP in the intestine of rme-1 mutant animals resembled the loss of retromer-dependent cargo MIG-14::GFP (abnormal cell migration-14) in rme-1 mutant animals (Fig. S2 D–F).

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). (A–G) 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). (L–O) 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 L–O. 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.

To test directly whether type I receptor SMA-6 recycling is dependent on the retromer pathway, we analyzed receptor localization in mutants lacking the core retromer subunit VPS-35 (vacuolar protein sorting factor-35) and several sorting nexins (SNX-1, SNX-3, and SNX-27) that may be specific for particular subsets of retromer-dependent cargo (24–27). vps-35 mutants and snx-3 mutants were severely defective in SMA-6 trafficking, whereas snx-1 mutants were mildly defective and snx-27 did not appear to affect SMA-6 (Fig. 2 A and C–F). Thus, SMA-6 is retromer-dependent and depends heavily on SNX-3, similar to known retromer cargo MIG-14/Wls (Wntless), a conserved membrane protein dedicated to the secretion of Wnt proteins. A key regulator specific to the ERC to plasma membrane recycling pathway is the small GTPase ARF-6. SMA-6 localization was unchanged in arf-6 deletion mutants, indicating the specificity of the requirement for retromer (Fig. 2 A and G).

Consistent with the idea that type II receptor DAF-4 recycles by a distinct mechanism, DAF-4 was not affected by loss of retromer core subunit VPS-35 (Fig. 2 L and O). Instead, we found that DAF-4::GFP accumulated in endosomes in arf-6 mutants (Fig. 2 L and N). Thus, DAF-4 is retromer-independent and ARF-6-dependent, the opposite of SMA-6.

If the receptor recycling pathways we identified for SMA-6 and DAF-4 are physiologically important for Sma/Mab signaling, we would expect that such signaling would be defective in recycling pathway mutants. To determine whether recycling of the type I and type II receptors is important for Sma/Mab signaling, we again assayed 3 outputs of Sma/Mab signaling in two epithelial tissue types, the hypodermis and intestine. We found that body size was strongly reduced in rme-1, vps-35, and arf-6 mutants, although not as severely as in mutants completely lacking the type I receptor SMA-6 (Fig. 2K). Furthermore, we found that in vps-35 and rme-1 mutants, hypodermal expression of the RAD-SMAD reporter and qRT-PCR analysis intestine-specific Sma/Mab target gene expression were reduced to levels similar to those found in mutants lacking the SMA-6 receptor, indicating the importance of receptor recycling to the ability of the cells to signal (Fig. 2 I and J). In addition, we found that in arf-6(tm1447), hypodermal expression of the RAD-SMAD reporter was reduced to levels similar to rme-1 and vps-35 mutants (Fig. 2Q). Taken together, our genetic and cell biological data demonstrate that distinct recycling pathways control the postendocytic itinerary of the type I and type II BMP receptors and that such recycling is critical to maintain cellular signaling capacity.

SMA-6 Is Mislocalized to the Lysosome in Retromer Mutants After Clathrin-Dependent Endocytosis.

To investigate our model further, we characterized the fate of SMA-6 in retromer mutants. We expected that SMA-6 levels were strongly reduced in retromer mutants because instead of recycling SMA-6, retromer mutants missort retromer-dependent cargo to the late endosome and lysosome (24, 28, 29). Indeed, we found that in wild-type cells, only 20% of SMA-6::GFP colocalized to the late endosome/lysosome marker tagRFP::RAB-7 (tag-red fluorescent protein::Rab GTPase-7), whereas 56% of SMA-6::GFP colocalized with tagRFP::RAB-7 in vps-35 mutants (Fig. 3). Furthermore, much of the remaining SMA-6::GFP signal remaining in vps-35 mutants appeared to be in the lumen of RAB-7-positive endosomes/lysosomes, whereas RAB-7 is restricted to the limiting membrane of these organelles. Thus, the 56% colocalization of SMA-6 with RAB-7 in vps-35 mutants likely represents an underestimate of SMA-6 missorting. As a further test of this model, we also used a genetic epistasis approach, blocking plasma membrane endocytosis or lysosome-mediated degradation, in a retromer-deficient vps-35 mutant. In a vps-35 mutant depleted of μ2-adaptin (DPY-23) by RNAi, SMA-6::GFP is not degraded and is trapped at the basolateral plasma membrane (Fig. 4 A–F). This indicates that retromer is not required for sorting SMA-6 until after its endocytosis from the plasma membrane. Furthermore, we found that in a vps-35 mutant depleted of CUP-5/mucolipin1 (coelomocyte uptake-defective-5), a protein required for lysosome function, the loss of SMA-6::GFP was blocked and, instead, SMA-6::GFP accumulated in the degradation-deficient late endosome/lysosome hybrid organelles characteristic of cup-5 mutants, and mildly at the plasma membrane (30) (Fig. 4 G–L). Thus, we also conclude that in a retromer mutant, postendocytic missorting sends SMA-6 to lysosomes, where it is inappropriately degraded.

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. (B–B″) 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.

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.

SMA-6 Binds Directly to the Retromer Complex.

Our results suggested that SMA-6 might be a direct target of the retromer sorting complex during its transit through endosomes after endocytosis. If this is true, we expected to find a physical interaction between the intracellular domain of SMA-6 and retromer. As a first test of this, we incubated lysates from C. elegans expressing GFP-tagged VPS-35 with beads containing immobilized SMA-6 intracellular domain purified from Escherichia coli as a GST fusion. GFP::VPS-35 protein was retained on the SMA-6-containing beads, but not by control beads containing GST alone (Fig. 5A). We next sought to determine whether such interaction was direct. We performed a similar assay using purified recombinant retromer cargo-selective complex (Vps35/Vps26/Vps29) and immobilized SMA-6 intracellular domain. VPS-35, VPS-26, and VPS-29 form a heterotrimer subcomplex of the retromer that mediates cargo recognition. The intracellular domain of the well-known retromer-dependent cargo protein, the cation-independent mannose-6-phosphate receptor (CI-MPR), was used as a positive control. SMA-6 pulled down the recombinant retromer cargo-selective complex in a similar manner to the CI-MPR positive control (Fig. 5B) (31). These results indicate that SMA-6 binds directly to retromer to mediate its intracellular sorting.

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.

Discussion

Members of the TGFβ superfamily of signal transduction pathways are conserved from early multicellular animals, such as trichoplax, to humans (32). Thus, our findings regarding the interplay of BMP receptor trafficking and signaling outputs have important implications for related receptors throughout metazoan phylogenies. Recently, two close vertebrate homologs of SMA-6, BMPRIA(ALK3) (bone morphogenetic protein type IA receptor/activin-like kinase 3) and ACVRIB(ALK4) (activin receptor type IB/activin-like kinase 4), were identified to be down-regulated in a proteomic study for cell-surface receptors altered by SNX27- and VPS35-depleted human HeLa cells (33). Although not investigated in individual detail, high-throughput proteomics suggested that ACVRIB was down-regulated in both SNX27- and VPS35-depleted cells, whereas BMPRIA was only down-regulated in SNX27-depleted cells. The cell surface proteome analysis identified only type I TGFβ superfamily receptors to be down-regulated. In contrast, no type II receptors were found to be down-regulated. A more distant homolog of SMA-6, TGFβR1(ALK5) (transforming growth factor-β receptor type I/activin-like kinase 5), was also suggested to be down-regulated in VPS35- and SNX27-depleted HeLa cells (33). Although a recent study failed to show a VPS35 RNAi effect in Madin–Darby canine kidney cells on TGFβR1(ALK5) (34), they did demonstrate that TGFβRII was mislocalized to both the basolateral and apical membrane, as opposed to its normal localization to the basolateral membrane. Examination of the role of the retromer complex on BMP signaling in Drosophila has been incongruent (27, 35, 36). On the basis of our genetic and cell biological data, as well as the preliminary data from the mammalian proteomic analysis, it is very likely that retromer-dependent regulation of type I BMP and Activin receptors is a conserved mechanism of TGFβ-receptor regulation.

Here we demonstrate that blocking receptor internalization, or receptor recycling, results in down-regulation of BMP signal transduction. This provides insight into how specific internalization and recycling pathways influence the molecular compartmentalization of the BMP receptors and provides insight into how altering this compartmentalization affects the signaling strength of the pathway. The identification of two distinct transport pathways for SMA-6 and DAF-4 during recycling of the receptors back to the plasma membrane suggests a mechanism by which aberrant signaling of these receptors can be avoided through physical disassociation of the active heteromeric complexes. Previously discovered differences in the rate of biosynthesis of the type I and II receptors were observed (37). Both the difference in rate of biosynthesis and the difference in trafficking, we report, may contribute to the difference in the half-life of the type I receptor, which has been identified to be longer than that of the type II receptor (37, 38).

In summary, our data demonstrate a novel function of the retromer in regulating BMP signaling through the regulation of a BMP type I receptors’ intracellular recycling. In addition, this regulation is unique to the type I receptor and did not affect the type II receptor in C. elegans, which we found traffics through an ARF-6-dependent recycling pathway. Taken together, our work shows the physiological importance of endocytosis and recycling to TGFβ signaling in the context of an intact developing organism and identifies a surprising mechanism to keep the type I and type II receptors apart as they depart the signaling endosome. We propose that this disparate recycling of the two receptors allows termination of signal transduction within the endosomal system while preserving both receptors for further rounds of signaling. Delineating the endocytic compartmentalization and pathways that regulate BMP signaling provides novel opportunities to characterize the effect of tumor-associated BMP receptor mutations on the compartmentalization of the receptors and in developing pharmacological inhibitors of BMP signaling in various diseases.

Materials and Methods

Full methods, including plasmid and transgenic strain construction, microscopy and image analysis, body size measurements, qRT-PCR analysis, protein expression and purification, and GST pull-down assay methods are found in SI Materials and Methods.

General Methods and Strains.

All C. elegans strains were derived originally from the wild-type Bristol strain N2. All strains were grown at 20 °C on standard nematode growth media plates. All C. elegans cultures, genetic crosses, and other husbandry were performed according to standard protocols (39).