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EVOLUTIONARY HOMOLOGS (part 2/2)

Type I TGF-beta and activin receptors

Information about the type I TGF-beta and activin receptors (Drosophila homolog: Baboon) is included because of their close relationship to the type I BMP receptor, but the receptors are different and should not be confused with one another.

Xenopus blastula cells activate different mesodermal genes as a concentration-dependent response to activin, which behaves like a morphogen. To understand how cells recognize morphogen concentration, radioactively labeled activin has been bound to cells and binding has been related to the choice of gene activation. The increasing occupancy of a single receptor type can cause cells to switch gene expression. Cells sense ligand concentration by the absolute number of occupied receptors per cell (100 and 300 molecules of bound activin induce Xbra and Xgsc, respectively, i.e., 2% and 6% of the total receptors) and not by a ratio of occupied to unoccupied receptors. The long duration of occupancy explains a previously described ratchet effect. These results suggest a new concept of morphogen gradient formation and interpretation that is particularly well suited to the needs of early development (Dyson, 1998).

An unexpected result of this work is that cells sense morphogen concentration and switch gene response when a remarkably small proportion of their receptors is occupied by ligand. However, this is very understandable if cells' interpretation of a morphogen gradient is envisioned in the following way: it is supposed that cells in one region of an embryo actively secrete morphogen for a few hours, during which time the concentration increases. After this, cells discontinue emitting morphogen, and its concentration decreases. It is believed that responsive cells monitor morphogen concentration continuously and respond by a ratchet mechanism to the highest concentration that they experience within their competent life. This proposed mechanism has at least two advantages for early development: (1) cells can bind ligand and respond rapidly to the morphogen and do not therefore need to wait for it to reach equilibrium. Through the ratchet effect, cells would always respond to the highest concentration that they experience within the few hours of their competent life even when the number of occupied receptors per cell might temporarily decrease, as during cell division. The ratchet effect would operate by the very high affinity of ligand for its receptors. (2) If cells were to respond at high occupancy but still measure the absolute numbers of occupied receptors, there could be some inconsistency of response due to titration of type I receptors. Under these conditions, the ligand type II receptor complexes first formed would see a much higher concentration of type I receptors than subsequent complexes. It would then be predicted that overexpression of Type I receptors would change response to activin concentration. The results presented in this paper show that this is not the case. It is concluded that, at the very low occupancies seen, both type I and type II receptors are in such excess that the few receptors actually used for signaling do not significantly reduce the overall pool of available receptors, and so increased occupancy can be directly reflected in increased signaling. The mechanism proposed can explain an apparent paradox. On the one hand, the ligand must be limiting in order to account for the concentration-dependent responses that are observed. On the other hand, the ligand must be in excess to be able to create a concentration gradient in distant cells. If this were not the case, most if not all the ligand would be sequestered by cells nearest the source, as may happen in the case of Hedgehog in Drosophila. This paradox may be explained as follows: it has been shown that cells can respond when very low levels of ligand are bound (100-300 molecules). This means that only a small proportion of the ligand in the intercellular space needs to be bound by receptors within the time available. In this way, cells are able to generate a concentration-dependent response without significantly reducing the concentration of ligand around them and therefore without disturbing the gradient (Dyson, 1998).

Activins and other ligands in the TGFbeta superfamily signal through a heteromeric complex of receptors. Disruption of signaling by a truncated type II activin receptor, XActRIIB (previously called XAR1), blocks mesoderm induction and promotes neuralization in Xenopus embryos. A type I activin receptor, XALK4 has been cloned and characterized. Like truncated XActRIIB, a truncated mutant (tXALK4) blocks mesoderm formation both in vitro and in vivo; moreover, an active form of the receptor induces mesoderm in a ligand-independent manner. Unlike truncated XActRIIB, however, tXALK4 does not induce neural tissue. This difference is explained by the finding that tXALK4 does not block BMP4-mediated epidermal specification, while truncated XActRIIB inhibits all BMP4 responses in embryonic explants. The neural/ectodermal fate engendered by BMP4 is likely to require another type I receptor AlK3 (BMPR1) and another type II receptor dedicated to ectodermal fate. Thus, the type I and type II activin receptors are involved in overlapping but distinct sets of embryonic signaling events (Chang, 1997).

ActRIB is a type I transmembrane serine/threonine kinase receptor that has been shown to form heteromeric complexes with the type II activin receptors to mediate activin signal. To investigate the function of ActRIB in mammalian development, both ActRIB-deficient mice and ES cell lines were generated by gene targeting. Analysis of the ActRIB-/- embryos shows that the epiblast and the extraembryonic ectoderm are disorganized, resulting in disruption and developmental arrest of the egg cylinder before gastrulation. To assess the function of ActRIB in mesoderm formation and gastrulation, chimera analysis was conducted. ActRIB-/- ES cells injected into wild-type blastocysts are able to contribute to the mesoderm in chimeric embryos, suggesting that ActRIB is not required for mesoderm formation. Primitive streak formation, however, is impaired in chimeras when ActRIB-/- cells contribute highly to the epiblast. Chimeras generated by injection of wild-type ES cells into ActRIB-/- blastocysts form relatively normal extraembryonic tissues, but the embryo proper develops poorly, probably resulting from severe gastrulation defect. These results provide genetic evidence that ActRIB functions in both epiblast and extraembryonic cells to mediate signals that are required for egg cylinder organization and gastrulation. Whereas the nodal receptor has yet to be identified through ligand binding analysis, the similar defects in primitive streak formation in nodal-/- and ActRIB-/- embryos raise the possibility that ActRIB might function as the type I receptor for nodal (Gu, 1998).

The TGFbeta family member activin induces different mesodermal cell types in a dose-dependent fashion in the Xenopus animal cap assay. High concentrations of activin induce dorsal and anterior cell types such as notochord and muscle, while low concentrations induce ventral and posterior tissues such as mesenchyme and mesothelium. Does this threshold phenomenon involve the differential effects of the two type I activin receptors ALK-2 and ALK-4? Injection of RNA encoding constitutively active forms of the receptors (here designated ALK-2* and ALK-4*) reveals that ALK-4* strongly induces the more posterior mesodermal marker Xbra (Drosophila homolog: Brachyenteron) and the dorsoanterior marker goosecoid in animal cap explants. Maximal levels of Xbra expression are attained using lower concentrations of RNA than are required for the strongest activation of goosecoid (Drosophila homolog: Goosecoid); at the highest doses of ALK-4*, levels of Xbra transcription decrease, as is seen with high concentrations of activin. By contrast, the ALK-2* receptor activates Xbra but fails to induce goosecoid to significant levels. At later stages, ALK-4* signaling induces the formation of a variety of mesodermal derivatives, including dorsal cell types, in a dose-dependent fashion; high levels also induce endoderm. By contrast, the ALK-2* receptor induces only ventral mesodermal markers. Consistent with these observations, ALK-4* is capable of inducing a secondary axis when injected into the ventral side of 32-cell stage embryos while ALK-2* cannot. Co-injection of RNAs encoding constitutively active forms of both receptors reveals that ventralizing signals from ALK-2* antagonize the dorsal mesoderm-inducing signal derived from ALK-4*, suggesting that the two receptors use distinct and interfering signaling pathways. Together, these results show that although ALK-2* and ALK-4* transduce distinct signals, the threshold responses characteristic of activin cannot be due to interactions between these two pathways; rather, thresholds can be established by ALK-4* alone. The effects of ALK-2* signaling are at odds with it behaving as an activin receptor in the early Xenopus embryo (Armes, 1997).

The type II receptors or TGFß and activin recognize these ligands free in the medium, whereas their type I receptors do not. The type I receptors recognize ligand-bound type II receptors, forming an oligomeric complex, probably a heterotetramer. The BMP receptor system is somewhat different. In this case, the type II receptors and the type I receptors both separately have low affinity for the ligand and together achieve high affinity binding. A central event in the generation of signals by type I and type II complexes is phosphorylation of the type I receptor. This is likely to be catalyzed by the type II receptor kinase. The activity of this kinase is required for phosphorylation in the cell, and it phosphorylates recominant type I receptor in vitro. Phosphorylation occurs in a cluster of five serine and threonine residues in the GS domain, a highly conserved region next to the N-terminus of the kinase domain in all type I receptors. The type II receptors have kinase activity that does not seem to be augmented by ligand binding. The ligand may be acting as an adaptor that brings a substrate-the type I receptor-to the primary receptor kinase (Massagué, 1996).

Activins and inhibins belong to the transforming growth factor (TGF-beta)-like superfamily and exert their effects on a broad range of cellular targets by modulating cell differentiation and proliferation. Members of this family interact with two structurally related classes of receptors (type I and type II), both containing a serine/threonine kinase domain. When expressed alone, the type II but not the type I activin receptor can bind activin. However, the presence of a type I receptor is required for signaling. For TGF-1, ligand binding to the type II receptor results in the recruitment and transphosphorylation of the type I receptor. Transient overexpression of the two types of activin receptors results in ligand-independent receptor heteromerization and activation. Nevertheless, activin addition to the transfected cells increases complex formation between the two receptors, suggesting a mechanism of action similar to that observed for the TGF-beta receptor. A stable cell line, expressing the two types of human activin receptors upon induction, was generated in the human erythroleukemia cell line K562. Activin specifically induces heteromer formation between the type I and type II receptors in a time-dependent manner. Activin signal transduction mediated through its type I and type II receptors results in an increase in the hemoglobin content of the cells and limits their proliferation. The inhibin antagonistic effects on activin-induced biological responses are mediated through a competition for the type II activin receptor but also require the presence of an inhibin-specific binding component (Lebrun, 1997).

Transforming growth factor-beta (TGFbeta) signaling requires phosphorylation of the type I receptor TbetaR-I by TbetaR-II. Although TGFbeta promotes the association of TbetaR-I with TbetaR-II, these receptor components have affinity for each other that can lead to their ligand-independent activation. The immunophilin FKBP12 (a cytosolic protein known to bind the immunosuppressants FK506 and rapamycin) binds to TbetaR-I and inhibits its signaling function. FKBP12 binding to TbetaR-I involves the rapamycin/Leu-Pro binding pocket of FKBP12 and a Leu-Pro sequence located next to the activating phosphorylation sites in TbetaR-I. Mutations in the binding sites of FKBP12 or TbetaR-I abolish the interaction between these proteins, leading to receptor activation in the absence of added ligand. FKBP12 does not inhibit TbetaR-I association with TbetaR-II; rather, it inhibits TbetaR-I phosphorylation by TbetaR-II. Rapamycin, which blocks FKBP12 binding to TbetaR-I, reverses the inhibitory effect of FKBP12 on TbetaR-I phosphorylation. By impeding the activation of TGFbeta receptor complexes formed in the absence of ligand, FKBP12 may provide a safeguard against leaky signaling resulting from the innate tendency of TbetaR-I and TbetaR-II to interact with each other (Chen, 1997).

A WD-40 repeat protein, TRIP-1, associates with the type II transforming growth factor beta (TGF-beta) receptor. Another WD-40 repeat protein, the Balpha subunit of protein phosphatase 2A, associates with the cytoplasmic domain of type I TGF-beta receptors. This association depends on the kinase activity of the type I receptor; it is increased by coexpression of the type II receptor (which is known to phosphorylate and activate the type I receptor) and allows the type I receptor to phosphorylate Balpha. Furthermore, Balpha enhances the growth inhibition activity of TGF-beta in a receptor-dependent manner. Because Balpha has been characterized as a regulator of phosphatase 2A activity, these observations suggest possible functional interactions between the TGF-beta receptor complex and the regulation of protein phosphatase 2A (Griswold-Prenner, 1998).

The effect of Balpha on the growth inhibition response of TGF-beta complements the role of Smads as effectors of TGF-beta receptor signaling. Smads function as transcriptional activators that induce the expression of various genes. Since the transcription of several genes is induced by Smads, Smads may induce growth inhibition by inducing transcription of the cdk inhibitors p15 and p21 in response to TGF-beta. Overexpression of Balpha induces growth inhibition to a level comparable to that of overexpression of Smads and, like the Smads, the effect of Balpha on growth inhibition depends on receptor activity. Furthermore, the antiproliferative effect of Balpha does not depend on Smad4, suggesting that TGF-beta receptor activation may induce two parallel pathways that lead to the antiproliferative response, one propagated by Smad proteins and the other one propagated through Balpha. Although the mechanism of the receptor-dependent growth inhibition by Balpha is not known, one possibility is that it acts through the ability of PP2A to regulate MAP kinase activity, especially since PP2A is a major enzyme involved in dephosphorylating MAP kinase. Therefore, altered PP2A activity following TGF-beta receptor activation might contribute to growth inhibition by deactivating this growth stimulatory pathway, theregy complementing the direct induction of growth inhibition by Smads. Moreover, a possible regulation of PP2A activity by TGF-beta may also directly affect the cell cycle, which would be consistent with the observed role of PP2A in cell cycle control (Griswold-Prenner, 1998 and references).

Two different chimeric TGF-beta superfamily receptors were generated: TbetaR-I/BMPR-IB, containing the extracellular domain of TGF-beta type I receptor (TbetaR-I) and the intracellular domain of bone morphogenetic protein type IB receptor (BMPR-IB), and TbetaR-II/ActR-IIB, containing the extracellular domain of TGF-beta type II receptor (TbetaR-II) and the intracellular domain of activin type IIB receptor (ActR-IIB). In the presence of TGF-beta1, TbetaR-I/BMPR-IB and TbetaR-II/ActR-IIB form heteromeric complexes with wild-type TbetaR-II and TbetaR-I, respectively, upon stable transfection in mink lung epithelial cell lines. TbetaR-II/ActR-IIB restores the responsiveness upon transfection in mutant cell lines lacking functional TbetaR-II with respect to TGF-beta-mediated activation of a transcriptional signal, extracellular matrix formation, growth inhibition, and Smad phosphorylation (see Drosophila Mothers against DPP). TbetaR-I/BMPR-IB and TbetaR-II/ActR-IIB forms a functional complex in response to TGF-beta and induced phosphorylation of Smad1. However, complex formation is not enough for signal propagation, which is shown by the inability of TbetaR-I/BMPR-IB to restore responsiveness to TGF-beta in cell lines deficient in functional TbetaR-I. The TGF-beta1-induced complex between TbetaR-II/ActR-IIB and TbetaR-I stimulates endogenous Smad2 phosphorylation, a TGF-beta-like response. This observation is in agreement with the current model for receptor activation, in which the type I receptor determines signal specificity (Persson, 1997).

In Mv1Lu cells the type I receptor TbetaRI mediates TGF-beta-induced gene expression and growth inhibition, while the closely related type I receptors Tsk7L and TSR1 are inactive in these responses. Using chimeras between TbetaRI and Tsk7L or TSR1, the structural requirements for TGF-beta signaling by TbetaRI have been defined. The extracellular/transmembrane or cytoplasmic domains of TbetaRI and Tsk7L are not functionally equivalent. The juxtamembrane domain, including the GS motif, and most regions in the kinase domain can functionally substitute for each other, but regions from kinase subdomains confer a distinct signaling ability. Replacement of these sequencez in TbetaRI by the corresponding domain of Tsk7L inactivates TGF-beta signaling, whereas its introduction into Tsk7L confers TGF-beta signaling. The differential signaling associated with this region is found in a sequence of eight amino acids, the L45 loop, which is exposed in the three-dimensional kinase structure and diverges highly between TbetaRI and Tsk7L or TSR1. Replacement of the L45 sequence in Tsk7L with that of TbetaRI confers TGF-beta responsiveness to the Tsk7L cytoplasmic domain in Mv1Lu cells. Thus, the L45 sequence between kinase subdomains IV and V specifies TGF-beta responsiveness of the type I receptor (Feng, 1997).

Interaction of Smad proteins with TGF-beta receptors

The Smad proteins function downstream of TGF-beta receptor serine/threonine kinases and undergo serine phosphorylation in response to receptor activation. Smad1 is regulated in this fashion by BMP receptors, and Smad2 and Smad3 by TGF-beta and activin receptors. BMP receptors phosphorylate and activate Smad1 directly. Phosphorylation of Smad1 in vivo involves serines in the carboxy-terminal motif SSXS. These residues are phosphorylated directly by a BMP type I receptor in vitro. Mutation of these carboxy-terminal serines prevents several Smad1 activation events, namely, Smad1 association with the related protein DPC4, accumulation in the nucleus, and gain of transcriptional activity. Similar carboxy-terminal serines in Smad2 are required for its phosphorylation and association with DPC4 in response to TGF-beta, indicating the general nature of the Smad activation process. As a direct physiological substrate of BMP receptors, Smad1 provides a link between receptor serine/threonine kinases and the nucleus (Kretzschmar, 1997).

Smad2 and Smad3 are structurally highly similar and mediate TGF-beta signals. Smad4 is distantly related to Smads 2 and 3, and forms a heteromeric complex with Smad2 after TGF-beta or activin stimulation. Smad2 and Smad3 interact with the kinase-deficient TGF-beta type I receptor (TbetaR)-I after it is phosphorylated by TbetaR-II kinase. TGF-beta1 induces phosphorylation of Smad2 and Smad3 in cultured Mv1Lu mink lung epithelial cells. Smad4 is found to be constitutively phosphorylated in Mv1Lu cells, the phosphorylation level remaining unchanged upon TGF-beta1 stimulation. Similar results are obtained using HSC4 cells, which are also growth-inhibited by TGF-beta. Smads 2 and 3 interact with Smad4 after TbetaR activation in transfected COS cells. In addition, TbetaR-activation-dependent interaction is observed between Smad2 and Smad3. Smads 2, 3 and 4 accumulate in the nucleus upon TGF-beta1 treatment in Mv1Lu cells, and show a synergistic effect in a transcriptional reporter assay using the TGF-beta-inducible plasminogen activator inhibitor-1 promoter. Dominant-negative Smad3 inhibits the transcriptional synergistic response by Smad2 and Smad4. These data suggest that TGF-beta induces heteromeric complexes of Smads 2, 3 and 4, and their concomitant translocation to the nucleus, which is required for efficient TGF-beta signal transduction (Nakao 1997).

Targets of TGF-beta receptors

TGF-beta initiates a signaling cascade leading to stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) activation. Expression of dominant-interfering forms of various components of the SAPK/JNK signaling pathways (including Rho-like GTPases (see Drosophila Rac1), mitogen-activated protein kinase (MAPK) kinase kinase 1 (MEKK1), MAPK kinase 4 (MKK4), SAPK/JNK, and c-Jun) abolishes TGF-beta-mediated signaling. Therefore, the SAPK/JNK activation most probably contributes to TGF-beta signaling (Atfi, 1997).

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