Endocytosis has long been considered a way of terminating signaling processes via receptor degradation. This was challenged in recent years when activated epidermal growth factor receptors and their effectors were found in what was considered to be the endosomal compartment . It is now evident that endomembrane structures serve as signaling platforms , and there are signaling endosomes or hermesomes which may be specialized for this process . The endomembrane system is divided into functionally and compositionally specialized subdomains [32,33], which determine the strength and duration of signaling responses by controlling recruitment of the downstream effectors of signaling complexes and sorting events such as recycling and transport to the lysosomal compartment for degradation. The endocytic pathway itself is controlled by signaling, demonstrating the extent to which signaling and trafficking are interlinked [34,35]. Furthermore, transport from early to late endocytic compartments is controlled by the cargo, and activated receptors may alter the kinetics to modulate their signaling duration .
Is internalization required for TGFβ family signaling?
The presence of SARA, Hrs and Endofin in early endocytic compartments questions whether signaling can occur from the plasma membrane or whether internalization is required to bring activated receptors to the endosome which is enriched in SARA and Endofin. This issue remains controversial, reflecting differences in experimental approaches and their limitations. TβRII undergoes constitutive internalization in the absence of ligand via clathrin-coated pits. This process is dependent on a short sequence (I218-I219-L220) that conforms to the di-leucine family of internalization signals [37,38] and the direct binding of type I and II receptors to β2-adaptin . No di-leucine motifs have been found in type I receptors. Interestingly, the NANDOR box is well conserved throughout type I receptors  and appears to play a role in type I receptor endocytosis.
Indeed, TβRI (ALK5) is internalized rapidly via CCVMR [41,42]. Ligand stimulation has no effect on the initial internalization rate or receptor recycling . Using a range of techniques including potassium (K+) depletion, which inhibits clathrin-mediated endocytosis , and a dominant-negative form of the dynamin GTPase, K44A dynamin II, which inhibits both clathrin- and caveolar-mediated endocytosis , various groups have addressed the requirement for internalization in TGFβ signaling. Lu et al.  found no involvement, however, several other groups have demonstrated the need for internalization [45,46].
Further studies showed that TGFβ receptors localize to both raft and nonraft membrane domains and the internalization route dictates whether signaling or degradation will ensue [11,47]. Internalization of TGFβ receptors, via the CCVMR, into an EEA1- and SARA-positive endosome promoted signaling. However, internalization via the raft–caveolar pathway, where Smad7 and Smurf2 are localized, promoted ubiquitin-dependent receptor degradation and inhibition of this pathway led to receptor stabilization, suggesting that trafficking of receptors to the SARA-positive early endosome functions to sequester receptors from the rafts and caveolae, thereby stabilizing the receptors .
In support of the above model, hyaluronan, an extracellular matrix polysaccharide, attenuated TGFβ signaling by increasing the segregation of TGFβ receptors into a lipid raft–caveolar compartment , whereas ADAM12 (a disintegrin and metalloproteinase) facilitated signaling by inducing the accumulation of TβRII in early endosomal vesicles and counteracting the internalization of TβRII into a caveolin1-positive compartment . Likewise, interleukin-6 augmented TGFβ signaling by increasing partitioning of TGFβ receptors to the nonlipid raft fraction (early endosomal) . No significant caveolar internalization was observed in the study by Mitchell et al. , in which nystatin (used at lower, more specific doses) had no effect on receptor internalization and degradation. Moreover, TGFβ receptors did not exhibit considerable co-localization in compartments positive for caveolin-1 .
What about the role of endocytosis in the signaling of other members of the TGFβ receptor family? With regard to activin A signaling, an ALK4 mutant, Alk4W477A, that was unable to undergo activin-dependent internalization, retained the ability to signal, demonstrating that ALK4 can signal without receptor internalization . However, in another detailed study addressing the memory of Xenopus embryonic cells to activin A exposure, the critical step in determining the duration of activin A signaling was the time spent by the ligand/receptor complexes in the endo-lysosomal pathway. Activin A internalization was required for correct signaling, suggesting that the localization of ligand to the endosomes was also required for a signaling step upstream of Smad2 activation. Dynamin-dependent endocytosis was necessary to generate signaling complexes, whereas delayed targeting to the lysosome ensured the persistence of signaling by such internalized complexes .
In agreement with the results with endosomal signaling of activin A/receptor complexes in Xenopus, work in Drosophila has shown that mutations in spinster (spin) , hrs/vps27p  and vps25 , which impair endosome-to-lysosome trafficking, cause an increase in BMP signaling, accompanied in some cases by increased levels of Thick Veins (an ortholog of ALK3/6). By contrast, Spichthyin (Spict), the Drosophila ortholog of the SPG6 and ichthyin protein family, which causes segregation (without degradation) of Wit (an ortholog of BMPRII) in early endosomes (Rab5-positive compartment), inhibits BMP signaling . Further work in Drosophila revealed that Nervous Wreck interacts with Thick Veins and the endocytic machinery to attenuate BMP signaling. Because Nervous Wreck co-localizes with Rab11, the authors suggested that Nervous Wreck might regulate the rate at which vacant Thick Veins receptors are recycled back to the plasma membrane following activation and internalization . Indeed, as mentioned below, TGFβ receptors are recycled via a Rab11-dependent mechanism independent of ligand binding, possibly as a means of rapidly and dynamically regulating surface receptor number and thus sensitivity to TGFβ . Recent biochemical data has shed more light on the link between BMP signaling and endocytic trafficking. BMPRI and BMPRII appear to be continuously internalized via CCVMR endocytosis, and BMPRII is also endocytosed via a caveolae- and cholesterol-dependent route . Smad1/5 phosphorylation seems to occur at the plasma membrane; however, continuation of Smad1/5-dependent signaling requires internalization via the CCVMR. The BMP receptor population that resides in cholesterol-enriched, detergent-resistant membrane fractions is required for Smad-independent BMP signaling . However, downregulation of caveolin-1 via siRNA resulted in a loss of BMP-dependent Smad phosphorylation and gene regulation, and was not linked only to Smad-independent signaling .
Rab GTPases are master regulators of vesicular transport and are distributed in distinct intracellular compartments. Rab5 is a key regulator of endocytosis that, by interacting with multiple effectors , regulates organelle-tethering, fusion and motility. Rab7 localizes to the late endocytic compartment and controls the trafficking of late endosomes . Therefore, conversion of Rab5 to Rab7 controls the progression of cargo from the early to the late endocytic compartment, but the cargo itself can also modulate the kinetics of this transport step . Thus, inputs from the Rab5/7 machinery or cargo (activated growth-factor receptors) may modulate the extent of downstream signaling by altering early/late endosome transport kinetics, thereby allowing activated receptors to access and/or reside for longer in an environment that allows productive signaling, especially in the case of TGFβ/activin A pathways in which SARA is enriched in the early endosome.
Indeed, RIN1, a Rab5 guanine exchange factor (GEF), via the activation of Rab5, directs TβRs into an endocytic pathway that promotes TGFβ signaling through Smads  (Fig. 1A). Silencing of RIN1, in turn, reduces TβR signaling efficiency. A negative feedback loop exists, whereby TβR signaling induces SNAI1, which in turn represses RIN1 expression. Interestingly, RIN1 promotes clathrin-dependent endocytosis of RTKs, such as MET and epidermal growth factor receptor, through direct binding to activated receptors and the stimulation of Rab5 proteins . This serves principally to direct RTK receptors to degradation, thereby leading to reduced signaling [63–65]. The differential signaling outcome of RTK versus TβR signaling by RIN1, however, suggests that Rab5-mediated endocytosis is not inextricably linked to a particular signaling outcome. Multiple endocytic complexes, each containing RIN1 and Rab5, and also other distinct components, may help explain different signaling outcomes during and following receptor internalization.
Figure 1. Control of TGFβ and activin/nodal receptor trafficking by small GTPases. (A) The role of Rab5 and Rab11 in TGFβ receptor endocytosis and recycling. The cycling of Rab5 between the GTP and GDP forms may influence the length and intensity of TGFβ/activin signaling cascades by regulating TGFβ–activin type I/II receptor trafficking via the early endocytic compartment. RIN1, a Rab5 GEF, via activation of Rab5, directs TβRs into an endocytic pathway that promotes TGFβ signaling through Smads. SNAI1, which represses RIN1 expression, is induced by TGFβ thus creating a negative feedback loop. Following clathrin-dependent internalization, TGFβ receptors recycle (irrespective of their activation state) in a Rab4-independent and Rab11-dependent manner. (B) The role of ARIP2, RalA and RalBP1 in activin A receptor internalization. ARIP2 interacts with ActRII and triggers their endocytosis via RalA/RalBP1 and POB1. POB1 interacts directly with the EH-containing proteins Epsin and Eps15. This protein complex acts as a scaffold to convey signals from the activin receptor to the endocytic machinery. (C) The role of Rap2 in activin/nodal receptor trafficking in Xenopus embryos. In the absence of ligand, Rap2 directs activin/nodal receptors into a Rab11-dependent recycling compartment, thereby avoiding degradation and maintaining cell-surface levels of receptors. Upon ligand addition, Rap2 competes with the Smad7/Smurf1 complex and delays receptor degradation, thus enhancing signaling.
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An additional consideration is the length of time TGFβ family receptors reside in early endosomes  once trafficked there. This is important because the signaling outcome is proportional to the residence time in this compartment. Trafficking of TGFβ family receptors via early endosomes with extremely fast kinetics will most likely have a minimal enhancing effect on signaling compared with early endosomal trafficking that is accompanied by blocking of further trafficking. Indeed, several studies on activin A and BMPs have revealed that the enhancing effect on signaling of various proteins was dependent on how long the relevant receptors resided on early endosomes [52–54]. Whether conversion of Rab5 to Rab7 or other mechanisms are responsible remains open. Our previous results suggest that Rab5 cycling between the GTP and GDP forms may influence the length and intensity of TGFβ/activin signaling cascades by regulating TGFβ–activin type I/II receptor trafficking via the early endocytic compartment . Indeed, in endothelial cells, Rab5S34N, a Rab5 mutant locked in the GDP form, caused augmented Smad3-dependent transcription in the absence of ligand. Because RN-tre, a specific Rab5 GTPase-activating protein (GAP) that blocks plasma membrane endocytosis, did not influence Smad3-dependent transcription, we concluded that the effect of Rab5S34N should have been the consequence of decreased degradative or recycling trafficking, leading to an accumulation of constitutively formed TGFβ–activin type I/II receptor complexes on early endosomal membranes.
Certainly, the station after early endosomes in the trafficking route of TGFβ family receptors is critical. Recycling back to the plasma membrane will influence signaling differently compared with trafficking towards late endosomes/lysosomes. This issue has been investigated by overexpressing dominant-negative forms of Rab4 (Rab4S22N) and Rab11 (Rab11S25N) and assessing TGFβ receptor trafficking . Rab4 regulates recycling from sorting/early endosomes to the plasma membrane, whereas Rab11 controls recycling through the perinuclear recycling endosomes  and trans-Golgi network to plasma membrane transport . Only Rab11S25N caused significant intracellular retention of TGFβ receptors, in both the presence and absence of ligand. Because co-localization of TGFβ receptors with Rab11 has been reported , it seems that, after clathrin-dependent internalization, TGFβ receptors recycle (irrespective of their activation state) in a Rab4-independent and Rab11-dependent manner (Fig. 1A). To date, the effect of Rab4 and Rab11 mutants or siRNA silencing on TGFβ signaling has not been investigated. However, it is expected to influence TGFβ signaling, especially its developmental aspects.
RalA/ Ral-binding protein 1
RalA is a multifunctional GTPase that is activated by receptor-activated Ras via recruitment of Ral GEFs [68–70]. Activated Ral associates with the Ral effector Ral-binding protein 1 (RalBP1), a cytosolic protein that is recruited to membranes following Ral activation  and activates hydrolysis of GTP bound to Rac1 and Cdc42. RalA has been implicated in many intracellular trafficking events  from the regulation of the endocytosis of EGF and insulin receptors  to secretion . Indeed, RalA, via its effector protein RalBP1, interacts with the μ2 subunit of the AP-2 complex  as well as with REPS1  and POB1  which are EGF receptor substrates containing Eps15 homology (EH) domains. POB1 interacts directly with the EH-containing proteins epsin and eps15, which have been reported to be involved in the regulation of EGF and transferin receptor endocytosis [67,78,79]. Thus, activation of RalA by EGF and insulin suggests that RalA/RalBP1 and its interactions with the μ2 chain of AP-2, REPS1, POB1, epsin and eps15 act as a scaffold that conveys signals from receptors to the endocytic machinery, thereby regulating ligand-dependent receptor-mediated endocytosis. Moreover, REPS1 interacts with Rab11-FIP2  a Rab11 effector that may couple REPS1-containing vesicles originating from clathrin-coated vesicles (and the early endocytic compartment) to the recycling endosomes.
RalA and RalBP1 appear to be involved in activin A receptor trafficking and signaling (Fig. 1B). It has been shown that activin receptor interacting protein 2 (ARIP2) interacts with ActRIIs and regulates their endocytosis via a PDZ domain-mediated interaction, concentrating them in a perinuclear compartment. Thus, ARIP2 reduces the response to ligands by decreasing the levels of ActRII at the plasma membrane . ARIP2 triggers the endocytosis of ActRIIs via Ral/RalBP1. Indeed, ARIP2 associates with ActRIIA and RALBP1 via its PDZ domain and C-terminal region, respectively. Because ARIP2C, the C-terminal deletion mutant of ARIP2 that does not bind RalBP1, failed to induce ActRII endocytosis, it appears that endocytosis of ActRIIs by ARIP2 is RalA/RalBP1 dependent. Moreover, activin A activates GDP–GTP exchange in RalA . Activation of RalA/RalBP1 by activin A is calcium dependent, in contrast to activation by EGF and insulin, which occurs via a Ras-dependent cascade . Interestingly, because only ActRIIs among all the serine/threonine kinase receptors for BMP/TGFβ/activin have the PDZ-binding sequence (ESSL for ActRIIA and ESSI for ActRIIB) , PDZ protein-regulated endocytosis and sorting is expected to influence only ActRIIs. Because ActRIIs bind both activins and also nodal and BMP7, ARIP2 is likely to play a role in shaping the activin/nodal/BMP gradient by regulating the endocytosis of ActRIIs.
Rap2 is a member of the Ras family of small GTPases whose effector domain is almost identical to that of Ras, and can therefore bind most Ras effectors. Rap2 inhibits many Ras pathways including Ras-induced Raf activation at the plasma membrane . Rap2 also binds to the Ral GEFs, Ral GDS, RGL and RLF . These proteins are also Ras effectors and induce nucleotide exchange leading to the formation of active RalA. As discussed above, Ral has been implicated in activin A receptor trafficking and may be linked to the molecular mode of action of Rap2 in Xenopus, as explained below.
In a very elegant study in Xenopus embryos, Rap2 was shown to regulate activin/nodal signaling by modulating receptor trafficking  (Fig. 1C). In the absence of ligand, Rap2 directs activin/nodal receptors into a Rab11-dependent recycling compartment, thereby avoiding degradation and maintaining cell-surface levels of receptors. Upon ligand addition, Rap2 no longer directs the receptors for recycling, but rather competes with Smad7 and delays receptor degradation, thus enhancing signaling. Moreover, Rap2 is initially enriched in the dorsal region of the blastulae, then as gastrulation proceeds, it decreases dorsally and increases ventrally. However, Smad7 is expressed uniformly across the dorso–ventral axis in early gastrulation and as gastrulation proceeds, Smad7 is restricted to the ventral region. Thus, Smad7 and Rap2 levels appear to regulate Smad2 activation along the dorso–ventral axis of the developing embryo.
Growing evidence links the progression of TGFβ receptor signaling to key regulatory steps in endocytic trafficking. These steps involve the active regulation of GDP-to-GTP exchange by various small GTPases of the Rab/Ral and Rap families. These mechanisms ensure optimal signal transduction from active receptor complexes to activated Smads.