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Molecular Endocrinology, doi:10.1210/me.2004-0079
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Molecular Endocrinology 18 (7): 1818-1826
Copyright © 2004 by The Endocrine Society

Receptor Internalization-Independent Activation of Smad2 in Activin Signaling

Yunli Zhou, Staci Scolavino, Sarah F. Funderburk, Laura F. Ficociello, Xun Zhang and Anne Klibanski

Neuroendocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Anne Klibanski, M.D., Massachusetts General Hospital, Neuroendocrine Unit, 55 Fruit Street, Bulfinch 457, Boston, Massachusetts 02114. E-mail: aklibanski{at}partners.org.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activin, a member of the TGFß family of cytokines, signals through heteromeric transmembrane complexes composed of type I and type II Ser/Thr kinase receptors. Activated by type II receptors, the type I receptor phosphorylates, thereby activating its effectors Smad2 and Smad3. It has been shown that the ligand-bound TGFß receptors endocytose to early endosomes, where they phosphorylate Smads. However, whether TGFß and activin can signal without receptor internalization is still in question. We report that a mutation changing Trp477 to Ala in the kinase domain rendered the type I activin receptor Alk4 unable to undergo ligand-dependent internalization. However, the resultant receptor, named Alk4W477A, retained the ability to phosphorylate Smad2 and mediate activin-induced transcription activation. Also, a Trp477 to Ala mutation abolished the endocytosis of Alk4T206D, a constitutively active type I activin receptor. The action of the mutant Alk4T206D became activin dependent. Finally, blocking endocytosis by depletion of intracellular potassium did not inhibit Smad2 phosphorylation by Alk4W477A. Taken together, our data indicate that activin receptors can transduce activin signals without endocytosis and suggest the possibility that an endocytosis-independent activin signaling pathway exists, which may act as an alternative mechanism for signal transduction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ACTIVIN IS A PLEIOTROPIC cytokine regulating a variety of biological and physiological processes. For example, activin stimulates production of pituitary FSH (1), promotes erythroid differentiation (2), and participates in regulation of bone metabolism (3), tissue repair (4), and inflammatory response (5). Activin signaling has been also linked to the development of human pituitary tumors (6, 7) and other human malignancies (8, 9). Activin belongs to the TGFß family of cytokines, including activin, TGFß, bone morphogenic proteins (BMPs) and Müllerian-inhibiting substance (MIS) in mammals (10). These cytokines transduce their signals through heteromeric transmembrane complexes composed of type I and type II Ser/Thr kinase receptors (11). In the presence of ligand, the type II receptor phosphorylates, thereby activating the type I receptor, which in turn activates the receptor-regulated Smads (R-Smads) by phosphorylation. The R-Smads in the activin and TGFß pathways are Smad2 and Smad3. The phosphorylated R-Smads form complexes with Smad4, which translocate into the nucleus and stimulate expression of the specific target genes (11). The phosphorylation of R-Smads by TGFß and activin receptors involves an adapter protein, SARA (Smad anchor for receptor activation) (12, 13), which recruits the unphosphorylated R-Smad to the activated receptor (12, 14). It has been shown that TGFß receptors form complexes with SARA and unactivated R-Smad at the plasma membrane, followed by internalization into early endosomes, where they phosphorylate Smad (15, 16). The receptor endocytosis involved in R-Smad activation is through clathrin-coated pits (15, 16, 17). Several studies have demonstrated that disruption of the clathrin-mediated endocytosis inhibits TGFß signaling (15, 16, 18), indicating that receptor internalization is critical in TGFß signal transduction. However, a study by Lu et al. (17) showed that blocking receptor endocytosis does not affect TGFß activation of Smad2, suggesting that TGFß can signal without receptor internalization. These apparently contradictory results may in part be due to the experimental approaches used to inhibit receptor endocytosis, including depletion of intracellular potassium, cell culture performed at low temperature, and expression of dominant-negative dynamin (16, 17, 18). Inhibition of endocytosis by these methods is not TGFß receptor specific and has broader effects on other cellular functions, which in turn, may affect TGFß signaling. Therefore, whether TGFß and activin can signal without receptor endocytosis needs to be further investigated by alternative approaches, which specifically target TGFß and activin signaling pathways.

Type II TGFß receptors contain an internalization signal belonging to the dileucine family (19), which is one of the motifs recognized by the clathrin-associated adapter complex AP2 (adaptor protein 2) (20). In addition, it has been reported that type II TGFß receptors interact with AP2 (21). Thus, it is likely that endocytosis of TGFß type II receptors is mediated by AP2. However, no dileucine motifs or other internalization signals have been found in type I receptors. Therefore, how type I receptors internalize remain elusive, although a weak interaction between TGFß type I receptors and AP2 has been reported (21). Interestingly, it has been reported that TGFß type I receptors carrying multiple amino acid mutations within a region near the carboxyl terminus fail to internalize and are unable to transduce signals (22). This region, referred to as NANDOR box (nonactivating-non-down-regulating), is well conserved throughout type I receptors for the TGFß family of cytokines including activin type I receptor Alk4 (22). Therefore, the NANDOR box plays a role in receptor endocytosis and type I TGFß family receptors may be actively involved in endocytosis of receptor complexes. To further investigate whether activin could signal without receptor internalization, we therefore performed a mutagenesis study with Alk4 by generating point mutations within and around the NANDOR box. Using this approach, we avoided the potential problems associated with the common methods used to interfere with clathrin-mediated receptor endocytosis. We found that one of the Alk4 mutants with a mutation changing Trp477 to Ala, named Alk4W477A, was unable to undergo activin-dependent internalization. However, it retained the ability to phosphorylate Smad2 and stimulate transcription from an activin-responsive promoter. Our data demonstrate that Alk4 can signal without receptor internalization, which may function as an alternative pathway in activin signaling.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Alk4 Carrying Trp477 to Ala Mutation Was Defective in Ligand-Dependent Internalization
To investigate whether activin can signal without receptor internalization, we substituted each individual amino acid residue with Ala from Met472 through Ser495 near the carboxyl terminus of kinase domain of Alk4, including the NANDOR box (Fig. 1AGo). The resultant mutant receptors were tagged with the FLAG epitope at their carboxyl termini and cloned into a mammalian expression vector pCI-neo. COS cells have been widely used in studies of TGFß/activin signal transduction (18, 19, 21, 23, 24, 25, 26). Therefore, we examined endocytosis of the mutant Alk4 receptors in COS1 cells by directly measuring the internalized receptor proteins, which were labeled with biotin on the cell surface. Each mutant Alk4 receptor was cotransfected with an expression vector for the myc-tagged ActRIIB into COS1 cells. After biotin labeling at 4 C, the transfected cells were moved to 37 C for activin treatment. We found that the wild-type (wt) Alk4 quickly internalized at 37 C even in the absence of activin, whereas activin treatment further enhanced its internalization (Fig. 1BGo), consistent with previous observations in TGFß type I receptors (17). We also found that the kinase-defective receptor Alk4KR internalized as the wt Alk4 (Fig. 1BGo, upper panel), indicating that kinase activity is not required for Alk4 endocytosis. We noted that changing a single amino acid within the NANDOR box did not affect receptor internalization (data not shown). Instead, a receptor carrying a mutation outside the NANDOR box changing Trp477 to Ala, named Alk4W477A, failed to internalize regardless of activin treatment (Fig. 1BGo, upper panel), suggesting that the Trp477 may play a critical role in endocytosis of type I activin receptors.



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  		Fig. 1. Alk4W477A Was Defective in the Ligand-Dependent Internalization

A, Mutagenesis studies with a single amino acid substitution were performed on the amino acid sequence between Met472 and Ser495 near the carboxyl terminus of Alk4. The boxed region is the NANDOR box (22 ). The Trp477 (W in bold) was substituted with Ala in the Alk4W477A mutant. B, Upper panel, Alk4W477A did not internalize in COS1 cells. The receptor internalization assay was performed as described in Materials and Methods. Cells were transfected with construct expressing FLAG-tagged Alk4W477A (W477A), wt Alk4 or kinase-deficient Alk4KR (KR). After being labeled with biotin at 4 C, the cells were treated without (–) or with (+) activin at 37 C. Biotins remained on cell surfaces were removed by glutathione reduction. The internalized receptor was precipitated with streptavidin beads and resolved on SDS-PAGE. Lower panel, Total FLAG-tagged receptor was detected in a duplicate set of cells, which received the same treatment as those for the internalization assay as described in Materials and Methods. Total, Total biotinylated activin receptors; Bkgd, cells were treated with glutathione before being moved to 37 C. C, Internalization of ActRIIB. The blot from (B) was stripped and reprobed with anti-myc antibody 9E10 to detect the myc-tagged ActRIIB. D, Alk4W477A forms complexes with ActRIIB. COS1 cells were transfected with pCI-Alk4W477A-FLAG or pCI-ALK4-FLAG and pcDNA-ActRIIB-myc. After labeled with [35S], the type I/II receptor complexes were isolated by two-step purification protocol (6 ) and resolved on SDS-PAGE. Type I and type II receptors were detected by autoradiography.

 
It has been shown that type II TGFß receptors endocytose constitutively (15, 19). Consistent with these reports, we found that ActRIIB internalized independent of activin treatment in cells expressing wt Alk4 or the kinase-defective Alk4KR (Fig. 1CGo). It has been suggested that the type I TGFß receptor internalizes through the ligand-mediated interaction with type II receptors (15). However, the internalized ActRIIB was readily detectable in cells expressing Alk4W477A (Fig. 1CGo), although no Alk4W477A internalization was observed in those cells (Fig. 1BGo). One possibility is that Alk4W477A does not form complexes with ActRIIB. To examine this possibility, we cotransfected COS1 cells with expression constructs for Alk4W477A-FLAG and ActRIIB-myc and isolated the type I/II receptor complexes using a two-step purification method (6). We found that complexes containing both Alk4W477A and ActRIIB were readily detected both in the presence and absence of activin treatment (Fig. 1DGo). These data strongly indicate that the type I activin receptor is also actively involved in the endocytosis of activin receptor complexes. Therefore, these findings indicate that the type I/II receptor complexes containing Alk4W477A do not internalize, and, the internalized ActRIIB observed in cells expressing Alk4W477A is mainly attributed to the free form of ActRIIB (Fig. 1CGo).

The kinase domain of the TGFß family receptors consists of eleven subdomains (27, 28, 29). Trp477 of Alk4 is located inside subdomain XI. If a Trp at this position is involved in receptor internalization, we reasoned that this Trp may also be conserved in other members of the TGFß family receptors. We aligned amino acid sequences of other TGFß type I receptors against those of Alk4 and found that a dipeptide sequence containing Cys476-Trp477 in Alk4 was conserved in all receptors examined, including those for TGFß (TßRI/Alk5) (30) and BMPs (BMPRIA and BMPRIB) (28, 31) (Fig. 2Go, upper panel), as well as those from Drosophila (Sax, Tkv, ATR-I) (32, 33, 34) and Caenorhabditis elegans (Daf-I) (35) (Fig. 2Go, upper panel). We also found that the type II TGFß family receptors conserve this dipeptide sequence of Cys-Trp in their kinase subdomain XI (Fig. 2Go, lower panel), including receptors for activin (ActRIIA and ActRIIB) (36, 37), TGFß (TßRII) (27), BMPs (BMPRII) (38), and MIS (AMHR, anti-Müllerian hormone receptor) (39). To investigate whether the conserved Cys was important in regulating receptor endocytosis, we substituted Cys476 with Ala in Alk4 and, surprisingly, found that the resultant receptor underwent endocytosis normally and was also functional in transducing activin signals (data not shown). These data indicate that only the conserved Trp may play a critical role in regulating internalization of TGFß family receptors.



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  		Fig. 2. A Trp in Kinase Subdomain XI Is Conserved in TGFß Family Receptors

Upper panel, Type I TGFß family receptors. ActR-IB, activin type I receptor; TßR-I, TGFß type I receptor; ActRI, a receptor binding to activin in vitro; BMPR-IA and BMPR-IB, BMP type I receptors; Tkv and Sax, Drosophila type I receptors for Dpp, functional homolog of mammalian BMPs; ART-I, a Drosophila type I receptor closely related to TßR-I and ActR-IB; Daf-1, C. elegans BMP receptors. Lower panel, Type II TGFß family receptors. ActR-IIA and ActR-IIB, Activin type II receptors; TßR-II, TGFß type II receptor; AHMR, type II receptor for anti-Müllerian hormone (also known as MIS); BMPR-II, BMP type II receptor; Punt, Drosophila type II receptor for Dpp.

 
Alk4 with Trp477 to Ala Mutation Was Functional in Activin Signaling
Smad2 is a phosphorylation target of Alk4 in activin signaling (40). To test the signal transduction function of Alk4W477A, we cotransfected pCI-Alk4W477A-FLAG and pcDNA-ActRIIB-myc with a FLAG-tagged Smad2 expression construct into COS1 cells and examined the phosphorylation status of Smad2 in the transfected cells. As expected, Smad2 was phosphorylated in cells expressing wt ALK4 (Fig. 3AGo), with more Smad2 phosphorylation in cells treated with activin than in cells not treated with activin (Fig. 3AGo); although no Smad2 phosphorylation was seen in cells expressing the kinase-defective Alk4KR (Fig. 3AGo). Interestingly, a significant amount of phosphorylated Smad2 was observed in activin-treated cells expressing Alk4W477A (Fig. 3AGo). However, the phosphorylated Smad2 was barely detectable in cells without activin treatment (Fig. 3AGo). Similar results were also observed in mink lung fibroblast L17 cells (Fig. 3BGo), which express very low levels of type I activin receptors. Thus, these data demonstrate that Alk4W477A is functional in transducing activin signals, although it is defective in ligand-dependent internalization.



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  		Fig. 3. Alk4W477A Was Functional in Transducing Activin Signals

A, Alk4W477A phosphorylated Smad2 in COS1 cells. The N-terminal FLAG-tagged Smad2 expression construct was cotransfected with plasmid expressing Alk4, Alk4KR, or Alk4W477A. After activin treatment, the FLAG-tagged Smad2 was immunoprecipitated by anti-FLAG antibody and resolved on SDS-PAGE. The phosphorylated Smad2 was probed with an anti-phospho-Smad2 antibody (upper panel). The blot was then stripped and reprobed with an anti-Samd2 antibody to detect total Smad2 protein (lower panel). B, Alk4W477A phosphorylated Smad2 in L17 cells. C, Alk4W477A stimulated transcription activation from the activin-responsive reporter construct 3TPLux. The assay was performed as previously described (6 ). Data are represented as mean ± SD for results from at least three independent experiments.

 
We found that activin treatment consistently induced more Smad2 phosphorylation in cells transfected with Alk4-FLAG than in those with Alk4W477A-FLAG, although the same amount of plasmid DNA for each construct was used for transfection (Fig. 3Go, A and B). To determine whether there is a functional difference in signal transduction between the wt Alk4 and Alk4W477A, we cotransfected 3TPLux, which contains an activin-responsive promoter controlling a cDNA encoding luciferase, with the same amount of Alk4 or Alk4W477A expression construct into L17 cells. The activin-induced transcription activation mediated by these receptors was evaluated by luciferase activities in cell lysates. As shown in Fig. 3CGo, the luciferase activity from activin-treated cells expressing Alk4W477A was about 50–60% of that from cells expressing the wt Alk4. There are at least two possibilities that may cause the decrease in signal transduction by Alk4W477A. One is that ligand-dependent receptor internalization is required for optimal activity of Alk4 in transducing activin signals. The other is that the mutation of Trp477 to Ala may affect other functions of the receptor in addition to internalization.

To exclude the possibility that the Smad2 phosphorylation by AlkW477A was due to receptor overexpression, we examined Smad2 phosphorylation in cells expressing various amount of Alk4W477A. We transfected COS1 cells with Alk4W477A or wt Alk4 expression construct at amounts ranging from 0.25–5 µg along with the fixed amount of pCI-FLAG-Smad2 and pcDNA-ActRIIB-myc. We found that transfection of 0.25 µg DNA, which expressed barely detectable wt Alk4 and Alk4W477A in COS1 cells, was enough to mediate activin-stimulated Smad2 phosphorylation (Fig. 4Go). Interestingly, when wt Alk4 expression increased, the ligand-independent Smad2 phosphorylation also increased significantly (Fig. 4AGo). In contrast, Smad2 phosphorylation in cells expressing Alk4W477A strictly depended on the activin stimulation regardless of the amount of receptor expressed in cells (Fig. 4BGo). This indicates that Alk4W477A is indeed a functional receptor in transduction of activin signals, and this endocytosis-independent signal transduction activity is ligand dependent.



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  		Fig. 4. Activin-Dependent Phosphorylation of Smad2 by Alk4W477A

A, Phosphorylation of Smad2 by wt Alk4 in COS1 cells. The DNA constructs expressing N-terminal FLAG-tagged Smad2 (2.5 µg) and ActRIIB-myc (1 µg) were cotransfected with plasmid expressing Alk4-FLAG at the indicated amount into COS1 cells. After activin treatment, the FLAG-tagged Smad2 was immunoprecipitated by anti-FLAG antibody and resolved on SDS-PAGE. The phosphorylated Smad2 was probed with an anti-phospho-Smad2 antibody (upper panel). The blot was then stripped and reprobed with an anti-FLAG antibody to detect total Smad2 protein (middle panel) and wt Alk4 (lower panel). B, Phosphorylation of Smad2 by Alk4W477A in COS1 cells. COS1 cells were transfected with FLAG-tagged Smad2 and Alk4W477A-FLAG as described above. The phosphorylated Smad2 was detected by an anti-phospho-Smad2 antibody (upper panel). The blot was stripped and reprobed with an anti-FLAG antibody to detect total Smad2 protein (middle panel) and Alk4W477A (lower panel).

 
Alk4T206D with Trp477 to Ala Mutation Failed to Internalize and Became Activin Dependent in Signaling
Alk4T206D is a constitutively active mutant type I activin receptor. It activates the downstream signaling cascade in the absence of activin (41). Similarly, we found that Alk4T206D phosphorylated Smad2 in COS1 cells and stimulated luciferase transcription from 3TPLux in L17 cells without activin treatment, and, the addition of activin did not affect the function of the receptor (Fig. 5Go, B and C). We also observed that Alk4T206D underwent endocytosis in the absence of activin (Fig. 5AGo). However, activin treatment enhanced internalization of Alk4T206D significantly (Fig. 5AGo), similar to that observed in wt Alk4 as seen in Fig. 1BGo, indicating that mutation at Thr206 does not affect receptor internalization.



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  		Fig. 5. Alk4TDW477A Did Not Internalize and Became Activin Dependent in Signaling

A, Alk4TDW477A did not internalize in COS1 cells (upper panel). COS1 cells were transfected with 2.5 µg of Alk4TDW477A or Alk4T206D plasmid. The receptor internalization was assayed as described in the legend for Fig. 1Go. The total FLAG-tagged receptor expression was determined in a duplicate set of the transfected cells (lower panel). P, Phosphorylated receptor. B, Alk4TDW477A phosphorylated Smad2 in COS1 cells. C, Alk4TDW477A stimulated luciferase expression from 3TPLux in L17 cells. Data are represented as mean ± SD for results from at least three independent experiments.

 
To examine how a defect in endocytosis affects the function of Alk4T206D, we substituted Trp477 with Ala in Alk4T206D and named the generated mutant receptor Alk4TDW447A. As expected, we found no internalization of Alk4TDW477A in COS1 cells transfected with pCI-Alk4TDW477A-FLAG regardless of activin treatment (Fig. 5AGo). However, it was surprising to find that Alk4TDW477A phosphorylated Smad2 mainly in cells treated with activin (Fig. 5BGo), whereas Alk4T206D phosphorylated Smad2 comparably in cells with and without activin treatment (Fig. 5BGo). Consistent with this observation, transcription activation from a 3TPLux reporter by Alk4TDW477A was also more dependent on activin treatment, whereas the transcription activation by Alk4T206D was not (Fig. 5CGo). These data further demonstrate that Trp477 is critical in endocytosis of Alk4 and the receptor endocytosis-independent activin signaling is ligand dependent.

Blocking Receptor Internalization by Potassium Depletion Did Not Affect Smad2 Phosphorylation by Alk4W477A
TGFß/activin receptors endocytose through a clathrin-mediated mechanism (16, 17). Depletion of intracellular potassium disrupts formation of the clathrin-coated pit, thereby inhibiting receptor internalization (16, 17). In agreement, we found that potassium depletion blocked internalization of Alk4 (Fig. 6AGo). As expected, Alk4W477A did not internalize either in the presence or absence of potassium (Fig. 6AGo). To investigate the effect of endocytic inhibition on signaling transduction by Alk4W477A, we examined Smad2 phosphorylation in potassium-depleted COS1 cells cotransfected with expression constructs for FLAG-Smad2 and Alk4W477A-FLAG. We found that the activin-induced phosphorylation of Smad2 by Alk4W477A in potassium-depleted cells was comparable to that in cells with potassium (Fig. 6BGo). This indicates that endocytosis inhibition does not affect the function of Alk4W477A, confirming that Alk4W477A functions independent of receptor internalization. Interestingly, we did not observe any decrease in Smad2 phosphorylation by wt Alk4 in cells with potassium depletion compared with that in cells with potassium (Fig; 6BGo). This result indicates that under our experimental condition wt Alk4 activates Smad2 without receptor internalization.



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  		Fig. 6. Depletion of Intracellular Potassium Did Not Affect Smad2 Phosphorylation by Alk4W477A

A, Potassium depletion abolished internalization of Alk4 in COS1 cells. Depletion of intracellular potassium was done as previously described (17 49 ). COS1 cells were transfected with expression constructs for Alk4 or Alk4W477A. The transfected cells were treated with hypotonic solution for 10 min, followed by isotonic treatment with solutions containing or lacking 10 mM KCl for 30 min at 37 C. Cells were then labeled with biotin at 4 C, followed by activin treatment at 37 C for 30 min, and the internalized receptor was detected as described in Materials and Methods. The lower panel represents the total FLAG-tagged receptor expression in the transfected cells from a duplicate experiment. B, Phosphorylation of Smad2 by Alk4 and Alk4W477A was not affected by potassium depletion (upper panel). COS1 cells were cotransfected with expression plasmids of FLAG-tagged Smad2 and Alk4 or Alk4W477A. After potassium depletion treatment as described above, cell lysates were prepared and the phosphorylated Smad2 was detected as described in Materials and Methods. The total Smad2 protein was detected by reprobing the stripped blot with an anti-Smad2 antibody (lower panel).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The clathrin-mediated endocytosis involves adapter complexes tethering the membrane-bound proteins to the clathrin-coated pits (42). One such adapter critical for internalization of many transmembrane receptors is AP2, composed of {alpha}, ß2, µ2, and {sigma}2 subunits (43). AP2 interacts with receptors by recognizing internalization signals, the best known of which are Tyr-based and dileucine-based motifs (43). TßRII has been found to contain a dileucine-based motif with the sequence of I218I219L220 (19), which is specifically recognized by the ß2 subunit of AP2 (43). Deletion of this motif blocks internalization of the receptor (19). Furthermore, AP2 has been found to interact with both TßRII and RßRI (21), although no internalization signals have been located in the type I receptor. Therefore, these data suggest that internalization of TGFß receptors, at least in part, is mediated by AP2.

Similar to TßRII, our data demonstrate that ActRIIB internalized constitutively (Fig, 1CGo). However, we did not find the dileucine motif in type II activin receptors by aligning amino acid sequences from ActRIIA and ActRIIB against those from TßRII (data not shown). Instead, Matsuzaki et al. (24) have reported that the endocytosis of activin type II receptors involves an activin receptor-interacting protein, PRIP2, which interacts with the last four amino acid residues of ActRIIA and ActRIIB, which are ESSL and ESSI, respectively. These data suggest that activin receptors may internalize by mechanisms that differ from those of TGFß receptors.

In contrast to the type II receptors, type I receptors for TGFß and activin undergo ligand-dependent endocytosis. Although an internalization signal motif is yet to be located in type I receptors, we found that substitution of Trp477 with Ala rendered Alk4 incapable of ligand-dependent internalization, despite the fact that the receptor formed complexes with ActRIIB (Fig. 1Go, B and D), suggesting that Alk4 may contain its own internalization signals and actively participate in the process of endocytosis. Our data also indicate that Trp477 plays an important role in regulating internalization of the receptor. In support of this possibility, we observed that all other type I and type II TGFß family receptors examined conserve this Trp at their kinase subdomain XI (Fig. 2Go). Furthermore, sequence alignment analysis revealed that a Trp at a similar position is also found in many membrane-bound receptors not belonging to the TGFß family, such as receptors for epidermal growth factor, platelet-derived growth factor, insulin, IGF-I, and macrophage colony-stimulating factor I (44), suggesting that this well-conserved Trp may play a more general role in participating in endocytosis of many transmembrane receptors. One possible function for Trp477 is that it is part of a novel internalization signal. Another possibility is that the location of this Trp is critical in maintaining proper protein conformation, which is required for signal recognition and/or interaction with adapters or other proteins necessary for receptor internalization.

Methods of endocytotic inhibition are commonly used to investigate the role of receptor internalization in TGFß signaling, including depletion of cellular potassium, overexpression of dominant-negative dynamin, and culturing cells at low temperature (16, 17, 18). Lu et al. (17) reported that potassium depletion and dominant-negative dynamin do not affect Smad2 phosphorylation by TGFß receptors. However, others have found that blocking receptor endocytosis by similar methods abolishes transduction of TGFß signals (15, 16, 18). Therefore, whether TGFß can signal without receptor internalization is still in question based on these studies. We think that the apparent contradictory conclusions reached by these reports are likely attributed to the experimental methods used in these studies. These methods block all clathrin-mediated endocytosis and have broader effects on other cellular functions, which may, in turn, affect TGFß signaling. In contrast, our study is based on the mutant activin receptor Alk4W477A, which was defective in receptor internalization (Fig. 1Go) and still had the ability to phosphorylate Smad2 in the presence of activin and mediated activin-induced transcription activation of a reporter gene (Figs. 3Go and 5Go). The inability to endocytose is an intrinsic part of Alk4W477A, which is not affected by endocytosis blocking methods used in previous studies. Therefore, our study provides direct evidence demonstrating that activin can signal in the absence of receptor internalization. Because Trp477 is conserved in TßRI (Fig. 2Go), our data predict that TßRI with Trp475 mutation is defective in internalization but remains functional in TGFß signal transduction.

One concern in a transient transfection experiment is protein overexpression. It is unlikely the case for Alk4W477A in our study. We examined Samd2 phosphorylation in COS1 cells with receptor expression at various levels. We observed that a significant amount of phosphorylated Smad2 was detected in activin-treated cells expressing a minimal amount Alk4W477A (Fig. 4Go). Similar results were also found in cells expressing low levels of wt Alk4. However, we noticed that high levels of wt Alk4 expression indeed caused activin-independent activation of Smad2 in COS1 cells (Fig. 4AGo), which is likely due to the self-activation by autophosphorylation (45). In contrast, Smad2 phosphorylation by Alk4W477A was strictly dependent on activin treatment regardless of protein levels of the receptor (Fig. 4BGo), indicating that the internalization-independent Smad2 phosphorylation by Alk4W477A is activin signaling pathway specific and not due to excessive expression of the receptor.

Alk4T206D is a constitutively active mutant. It endocytosed similar to the wt receptor (Figs. 1BGo and 5AGo). We noticed that much more receptor internalized in cells treated with activin than in cells without treatment (Fig. 5AGo). However, the Smad2 phosphorylation by Alk4T206D was not affected by activin treatment (Fig. 5BGo). Apparently, the function and receptor internalization in Alk4T206D does not completely correlate. This raises the possibility that Alk4T206D phosphorylates Smad2 and activates transcription from 3TPLux, at least partly, in the absence of receptor endocytosis. Interestingly, Alk4T206D with Trp477 to Ala mutation became activin dependent in phosphorylation of Smad2 and activation of transcription from reporter 3TPLux (Fig. 5Go, B and C). It is generally believed that substitution of Thr206 with Asp mimics the phosphorylation of the receptor, meaning that Alk4T206D has an active conformation that is usually achieved by activin-induced phosphorylation at the glycine- and serine-rich domain of the receptor. It is likely that this active conformation was altered when Trp477 was changed to Ala, resulting in a receptor with a conformation closer to wt Alk4. This may account for the activin-dependent activities of Alk4TDW477A. In supporting this possibility, a large slow migrating band was shown in lanes with Alk4T206D when the total receptor expression was examined as a control for the endocytosis assay (Fig. 5AGo, arrow P in lower panel). The slow migrating band is presumed to be the phosphorylated receptor as demonstrated in our previous study (6), which may be the result of autophosphorylation by the constitutively active Alk4T206D. In contrast, no such bands were seen in lanes with Alk4TDW477A (Fig. 3AGo, lower panel).

Receptor endocytosis in TGFß signal transduction has been well investigated, although studies on its role in activin signaling are not established. Although it is assumed that activin signals through similar mechanisms to TGFß, conclusions obtained from studying TGFß signaling pathway are not completely applicable to activin signaling. For example, TßRII contains a dileucine internalization signal motif (19), which is likely used by AP2 to mediate endocytosis of the receptor (21). In contrast, ActRIIB does not have this motif, suggesting that its endocytosis may not involve AP2. Most studies have demonstrated that receptor endocytosis is critical in transducing TGFß signals. Although our data do not exclude the important role that the receptor internalization may play in activin signaling, they do suggest the existence of an endocytosis-independent pathway for activin signal transduction, which may act as an alternative mechanism to endocytosis-dependent activin signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture and Plasmid Constructs
COS1 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). L17 cells were kindly provided by Dr. J. Massague (Memorial Sloan-Kettering Cancer Center, New York, NY) (41). Expression constructs for the FLAG-tagged wt Alk4 (pCI-Alk4-FLAG) and the myc-tagged ActRIIB (pcDNA3RIIB-myc) have been described previously (6). pCI-Alk4W477A-FLAG was constructed using a PCR-based mutagenesis method, which has been described previously (46). To construct pCI-ALK4TD-FLAG, an Alk4 cDNA fragment containing Thr206 to Asp mutation was isolated from pCMV5B/RIB-HA-T206D (kindly provided by Dr. J. Massague) and inserted into pCI-Alk4-FLAG to replace the corresponding wt cDNA sequences. pCI-Alk4TD-W477A-FLAG was similarly constructed to contain Thr206 to Asp mutation in Alk4W477A. pCI-Alk4KR-FLAG, expressing a kinase-deficient Alk4, was constructed according to a previously published report (41). The activin-responsive reporter construct, 3TPLux, was kindly provided by Dr. J. Massague (47). A full-length cDNA encoding human Smad2 (IMAGE Consortium CloneID 4449540) was obtained from Open Biosystems (Huntsville, AL). A DNA fragment encoding the FLAG epitope was fused to the 5'-end of the Smad2 coding region by PCR to generate a FLAG-tagged Smad2 expression construct, pCI-FLAG-Smad2.

Receptor Internalization
Receptor internalization assays were performed as previously described (17, 48). Briefly, COS1 cells were grown in 60-mm dishes and transfected with 2 µg Alk4 expression construct plus 2 µg of pcDNA3RIIB-myc using a TransIT LT1 reagent according to the manufacturer’s instructions (Takara Mirus Bio, Madison, WI). Cells were biotinylated with EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford, IL) at 4 C. After the transfected cells were treated with 2 nM activin A for 30 min at 37 C, biotins remaining on cell surfaces were removed by glutathione reduction. Cells were then lysed with lysis buffer containing 1% Triton X-100. The biotinylated receptor was precipitated with streptavidin beads (Pierce) and resolved on 10% SDS-PAGE. The type I receptors were probed with the monoclonal anti-FLAG antibody M2 (Sigma-Aldrich, St. Louis, MO). The blot was then stripped and reprobed with an anti-myc antibody (9E10; Santa Cruz Biotechnology, Santa Cruz, CA) to detect the biotinylated ActRIIB. To detect total Alk4 expression, a duplicate experiment was performed except that no NHS-SS-Biotin was included in the biotinylation reaction. The FLAG-tagged receptor was precipitated with M2 antibody and detected with a rabbit anti-FLAG antibody (Sigma-Aldrich).

Smad2 Phosphorylation and Luciferase Assays
Luciferase assays in L17 cells transfected with 3TPLux and Alk4 receptors were performed as previously described (6). For the Smad2 phosphorylation assay, COS1 or L17 cells were seeded into 100-mm dishes. For COS1 cells, pCI-FLAG-Smad2 (2.5 µg) plus 1 µg or the amount as indicated of each Alk4 expression construct and 1 µg of pcDNA3RIIB-myc was transfected using TransIT-LT1 reagent in Opti-MEM (Invitrogen Life Technologies, Carlsbad, CA). For L17 cells, the same amount of pCI-FLAG-Smad2 and 5 µg Alk4 expression construct was transfected using LipofectAmine 2000 reagent (Invitrogen Life Technologies). Twenty-four hours later, cells were treated with 2 nM activin A in serum-free DMEM for 1 h at 37 C and subsequently lysed with RIPA lysis buffer. The FLAG-tagged Samd2 was immunoprecipitated with 3 µg of M2 antibody and resolved on 8% SDS-PAGE. The phosphorylated Smad2 was detected with a phospho-Smad2 (Ser465/467) antibody from Cell Signaling (Beverly, MA; catalog no. 3101). The blot was then stripped and reprobed with anti-Smad2 antibody (Santa Cruz Biotechnology) to detect total FLAG-tagged Smad2.


   FOOTNOTES
 
This work was supported by F32 CA88519 (Y.Z.) and R01-DK-40947 from the National Institutes of Health, and the Jarislowsky Foundation.

Abbreviations: AP2, Adaptor protein 2; BMP, bone morphogenic protein; MIS, Müllerian-inhibiting substance; NANDOR box, nonactivating-non-down-regulating; R-Smads, receptor-regulated Smads; wt, wild-type.

Received for publication February 25, 2004. Accepted for publication April 6, 2004.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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