ß-Arrestin 2 Mediates Endocytosis of Type III TGF-ß Receptor and Down-Regulation of Its Signaling

See allHide authors and affiliations

Science  05 Sep 2003:
Vol. 301, Issue 5638, pp. 1394-1397
DOI: 10.1126/science.1083195


β-Arrestins bind to activated seven transmembrane–spanning (7TMS) receptors (G protein–coupled receptors) after the receptors are phosphorylated by G protein–coupled receptor kinases (GRKs), thereby regulating their signaling and internalization. Here, we demonstrate an unexpected and analogous role of β-arrestin 2 (βarr2) for the single transmembrane–spanning type III transforming growth factor–β (TGF-β) receptor (TβRIII, also referred to as betaglycan). Binding of βarr2 to TβRIII was also triggered by phosphorylation of the receptor on its cytoplasmic domain (likely at threonine 841). However, such phosphorylation was mediated by the type II TGF-β receptor (TβRII), which is itself a kinase, rather than by a GRK. Association with βarr2 led to internalization of both receptors and down-regulation of TGF-β signaling. Thus, the regulatory actions of β-arrestins are broader than previously appreciated, extending to the TGF-β receptor family as well.

The family of transforming growth factor–β ligands (TGF-β1, -β2, -β3) regulates cellular processes by binding to heteromeric complexes of high-affinity single transmembrane–spanning type I (TβRI), type II (TβRII), and type III (TβRIII or betaglycan) TGF-β receptors (1). Upon ligand binding, TβRII, a constitutively active serine-threonine kinase, recruits and phosphorylates TβRI, thereby activating a serine-threonine kinase activity of TβRI. The activated TβRI then recruits and phosphorylates Smad transcription factors, which translocate to the nucleus to regulate TGF-β–responsive genes.

Whereas TβRII binds only TGF-β1 and TGF-β3 independently, TβRIII binds all three TGF-β isoforms with high affinity and, particularly in the case of TGF-β2, presents this ligand to TβRII (2). However, in vivo, TβRIII has been shown to be required not only for TGF-β2 signaling (3), but also for TGF-β1 signaling (4). Another important property of TGF-β receptors is that they are constitutively internalized, and this internalization may have important roles in TGF-β signaling (58).

β-Arrestins bind to seven transmembrane–spanning (7TMS) receptors after they are phosphorylated by G protein–coupled receptor kinases (GRKs) (9, 10). β-Arrestin binding serves to uncouple the receptors from the G proteins, functionally link the receptors to other signaling pathways, including the mitogen-activated protein kinase cascades (9), and to mediate their internalization by clathrin-coated pits (11).

β-Arrestins and green fluorescent protein (GFP)–tagged β-arrestins, when expressed alone, are diffusely distributed throughout the cytoplasm (12). However, when they are expressed together with 7TMS receptors, agonist stimulation leads to their translocation to the plasma membrane, where they remain localized with the receptors or rapidly internalize with the receptors and colocalize with them in intracellular vesicles (12, 13). To investigate whether β-arrestins have a role in regulating TGF-β superfamily receptors, we coexpressed the type I (ALK-1–ALK-6), type II (activin, bone morphogenetic protein, TGF-β), and type III (TβRIII) TGF-β superfamily receptors with βarr2-GFP in human embryonic kidney (HEK) 293 cells and assessed the localization of βarr2-GFP. βarr2-GFP alone was diffusely expressed in the cytoplasm (Fig. 1A). This diffuse distribution was unaffected by coexpression of any of the type I or type II TGF-β superfamily receptors (Fig. 1, B and C) (14), but expression of TβRIII resulted in redistribution of βarr2-GFP into intracellular vesicles (Fig. 1D). TGF-β1 stimulation for up to 16 hours did not enhance the TβRIII-mediated redistribution of βarr2-GFP into intracellular vesicles, nor did it result in TβRI-mediated or TβRII-mediated redistribution of βarr2-GFP (Fig. 1, B and C) (14). TβRIII-mediated redistribution of βarr2-GFP into intracellular vesicles was specific because distribution of βarr1-GFP was not affected by coexpression of TβRIII (fig. S1). Interaction of β-arrestins with 7TMS receptors generally results in receptor internalization (11). TβRIII was localized primarily at the plasma membrane when expressed alone (Fig. 1E). However, coexpression with βarr2-GFP led to nearly complete internalization of TβRIII into intracellular vesicles, where TβRIII colocalized with the βarr2-GFP (Fig. 1, F to H).

Fig. 1.

Recruitment and colocalization of βarr2 with TβRIII. (A to D) Confocal images of βarr2-GFP expressed alone (A) or with Myc-TβRI(B), His-TβRII (C), or hemagglutinin (HA)–TβRIII (D) in HEK293 cells. (E) A confocal image of HA-TβRIII in HEK293 cells. (F to H) Confocal images of HA-TβRIII (F) and βarr2-GFP (G) in the same HEK293 cell overexpressing HA-TβRIII and βarr2-GFP. βarr2-GFP colocalizes with HA-TβRIII in intracellular vesicles in the merged image (H) (yellow vesicles). Scale bar, 10 μm. Data are representative of three independent experiments.

Deletion of TβRIII's short cytoplasmic domain (TβRIIIΔcyto) completely abrogated redistribution of TβRIII with βarr2-GFP (14). TβRIIIΔ3, which lacks the class I PDZ binding motif that mediates interaction of TβRIII with the PDZ domain–containing protein GAIP interacting protein, C terminus (GIPC) (Fig. 2A) (15), recruited βarr2-GFP as well as did full-length TβRIII (Fig. 2, B and C). However, TβRIIIΔ10, which lacks the last 10 residues of the cytoplasmic domain, did not interact with βarr2-GFP (Fig. 2D), localizing the βarr2-interacting domain to the seven amino acid residues that differ between these mutants (Fig. 2A). This serine-threonine–rich sequence resembles β-arrestin–interacting domains in 7TMS receptors (16). To further localize the interaction, each serine and threonine in this sequence was individually mutated to alanine. All of the serine mutants interacted with βarr2-GFP as well as did wild-type TβRIII (14). However, mutation of Thr841 to Ala in TβRIIIT841A (Fig. 2A) completely abolished interaction with βarr2-GFP (Fig. 2, E to H). The βarr2-TβRIII interaction, the site of interaction, the lack of ligand effect, and the specificity of the interaction for βarr2 were further confirmed by cellular coimmunoprecipitation studies (figs. S2 to S4).

Fig. 2.

Mapping of the interaction site of TβRIII with βarr2. (A) Schematic representation of wild-type, COOH-terminal truncation mutants, and the T841A point mutation mutant of HA epitope–tagged TβRIII. ECD, extracellular domain; TM, transmembrane domain; RIII, TβRIII. (B to E) HEK293 cells transiently expressing βarr2-GFP and HA epitope–tagged wild-type or mutant TβRIII, as indicated, were directly visualized for βarr2-GFP localization by confocal microscopy. (F to H) HEK293 cells transiently expressing βarr2-GFP and HA epitope–tagged mutant TβRIIIT841A were fixed, permeabilized, and stained with antibody to HA to visualize TβRIIIT841A localization. Overlay image (H) depicts lack of colocalization of βarr2-GFP with TβRIIIT841A. Scale bar, 10 μm. Data are representative of three independent experiments.

The cytoplasmic domain of TβRIII is rich in serine and threonine residues (Fig. 2A) and is phosphorylated in vivo by TβRII (17). Expression of TβRIIIΔgag (which cannot be modified by glycosaminoglycan chains and thus runs as a discrete band) with TβRII in [32P]orthophosphate-labeled HEK293 cells resulted in phosphorylation of TβRIII (fig. S5A), whereas expression with TβRII-K277R (catalytically inactive), GRK2, or GRK5 had no effect on phosphorylation of TβRIII (fig. S5A). In contrast, TβRIIIΔgagT841A was phosphorylated at markedly lower levels by TβRII in vivo (fig. S5B), identifying Thr841 as a major site of phosphorylation. In the absence of a coexpressed kinase or in the presence of coexpression of GRK2 or TβRII-K277R with TβRIII, 30 ± 5% of cells expressing βarr2-GFP demonstrated recruitment of βarr2-GFP to intracellular vesicles. However, coexpression of TβRII with TβRIII increased the percentage of cells with recruitment of βarr2-GFP to intracellular vesicles to 80 ± 8%. As expected, coexpression of TβRII with TβRIIIT841A had little or no effect. The ability of TβRII (and not a TβRII kinase dead mutant) to increase the βarr2-TβRIII interaction while having no effect on TβRIIIT841A was further confirmed by cellular coimmunoprecipitation studies (fig. S2). Although βarr2 does not interact with TβRII (Fig. 1C) or promote the internalization of TβRII (Fig. 3, A to C), TβRII is known to form complexes with TβRIII in vivo (2). Indeed, in the presence of TβRIII, TβRII cointernalized and colocalized with the TβRIII/βarr2-GFP complexes in intracellular vesicles (Fig. 3, D to F). The colocalization of these receptors with βarr2-GFP in vesicles was the result of internalization from the cell surface as an antibody to TβRIII applied to the outside of the cells also colocalized with the complex in endocytic vesicles (fig. S6). Taken together, these results suggest that TβRII phosphorylates TβRIII on Thr841 to mediate recruitment of βarr2 to TβRIII and internalization of TβRIII and TβRII in vivo.

Fig. 3.

TβRII cointernalizes with TβRIII and βarr2. HEK293 cells transiently expressing βarr2-GFP and His epitope–tagged TβRII (A to C) or βarr2-GFP, His epitope-tagged TβRII, and HA epitope–tagged TβRIII (D to F) were fixed, permeabilized, and stained to visualize epitope-tagged TβRII localization. (A to C) Confocal images of His-TβRII (A) and βarr2-GFP (B) in the same HEK293 cell overexpressing His-TβRII and βarr2-GFP. βarr2-GFP does not colocalize with His-TβRII in the merged image (C). (D to F) Confocal images of His-TβRII (D) and βarr2-GFP (E) in the same HEK293 cell. βarr2-GFP colocalizes with His-TβRII in the presence of TβRIII (F) (merged image, yellow vesicles). Scale bar, 10 μm. Data are representative of three independent experiments.

To establish a role for βarr2 in regulating endogenous TGF-β receptors, we used small interfering RNA (siRNA) to reduce the amounts of endogenous βarr2 expression in HEK293 cells, which express endogenous TβRIII, TβRII, and TβRI (14, 18), and monitored internalization of 125I–TGF-β1 by these endogenous receptors. siRNA directed against βarr2 specifically and potently decreased βarr2 expression (by ∼90% relative to control cells) while having essentially no effect on the expression of βarr1 (Fig. 4A). This reduction in βarr2 concentrations was associated with an 80% reduction in the internalization of 125I–TGF-β1 relative to that in cells treated with control siRNA (Fig. 4B), but no alteration in steady-state levels of cell surface TβRIII and TβRII (14).

Fig. 4.

βarr2 mediates internalization of TGF-β1 and down-regulation of TGF-β signaling. (A) Cell lysates from HEK293 cells transfected with control siRNA (left lane) or siRNA against βarr2 (right lane) were immunoblotted with antibody to β-arrestin (A1CT). (B) TGF-β1 internalization was determined by radioligand binding on siRNA-treated HEK293 cells. Data are expressed as the percent internalization, calculated as [(cpm of internalized receptor/cpm of total cell binding) × 100], where cpm is counts per minute. The results shown are the mean ± SEM of three independent experiments performed in triplicate. *P < 0.05 (unpaired t test). (C) Primary keratinocytes from newborn βarr2 littermate wild-type (WT), heterozygote (Het), or knockout (KO) pups were assayed for TGF-β1– or TGF-β2– (100 pM) mediated growth inhibition after 48 hours of treatment by measurement of [3H]thymidine incorporation. Data are expressed as the percent growth inhibition relative to untreated control cells. The results shown are the mean ± SEM of three independent experiments performed in triplicate. *P < 0.05 (unpaired t test) compared to the response of wild-type keratinocytes.

Studies have reported both clathrin-dependent (5, 6) and -independent mechanisms (8, 19) for endocytosis of TGF-β receptors. Consistent with the results obtained here, these studies demonstrated that the internalization of TGF-β receptors is ligand independent (7, 8, 2022). Moreover, direct interaction of TβRII with AP-2 in the clathrin-mediated pathway has also been reported, suggesting that not all TGF-β receptor internalization is mediated by the TβRIII–β-arrestin interaction reported here (22).

Could βarr2-mediated internalization of TβRIII/TβRII complexes serve to down-regulate TGF-β signaling? To test this hypothesis, we used keratinocytes derived from βarr2 knockout mice (23). Keratinocytes express TβRIII, TβRII, and TβRI, which mediate the antiproliferative effects of TGF-β on these cells (24, 25). Keratinocytes were isolated from homozygous (βarr2/), heterozygous (βarr2+/), and wild-type (βarr2+/+) littermate control newborn mice, and cellular proliferation was assessed by [3H]thymidine incorporation in the presence or absence of TGF-β (Fig. 4C). Treatment of keratinocytes with TGF-β1 or TGF-β2 (100 pM) for 48 hours revealed progressively increased sensitivity to TGF-β–mediated inhibition of proliferation as βarr2 expression decreased from wild-type levels, to heterozygous levels, to homozygous knockout levels (Fig. 4C). The regulatory effect of βarr2 was more dramatic for TGF-β2– than TGF-β1–induced signaling, as would be expected from the specific requirement of TGF-β2 for TβRIII. That eliminating βarr2 expression in vivo enhances TGF-β signaling strongly supports a role for βarr2 in down-regulating TGF-β signaling.

Our findings indicate a physiologically significant, unanticipated role for βarr2 in regulating TGF-β signaling through internalization and subsequent decreased signaling of TGF-β receptors. These studies extend the range of receptors with which β-arrestins can interact, expand the scope of potential kinases that can target β-arrestin to the cytoplasmic domain of receptors, and demonstrate that β-arrestin binding to one receptor, TβRIII, can lead to the cointernalization of another receptor, the kinase TβRII.

Supporting Online Material

Materials and Methods

Figs. S1 to S6.


References and Notes

View Abstract

Stay Connected to Science

Navigate This Article