Transforming growth factor-␤ (TGF-␤) binds to and signals via two serine-threonine kinase receptors, type I (T␤RI) and type II (T␤RII). The oligomerization of TGF-␤ receptors modulates ligand binding and receptor trafficking and may contribute to signal diversification. However, numerous features of the molecular domains that determine the homo-and hetero-oligomerization of full-length receptors at the cell surface and the mode of these interactions remain unclear. Here, we address these questions through computerized immunofluorescence co-patching and patch/fluorescence recovery after photobleaching measurements of different combinations of epitopetagged receptors and their mutants in live cells. We show that T␤RI and T␤RII are present on the plasma membrane both as monomers and homo-and hetero-oligomers. The homodimerization of T␤RII depends on a cytoplasmic juxtamembrane region (amino acid residues 200 -220). In contrast, the cytoplasmic domain of T␤RI is dispensable for its homodimerization. T␤RI⅐T␤RII hetero-oligomerization depends on the cytoplasmic domain of T␤RI and on a C-terminal region of T␤RII (residues 419 -565). TGF-␤1 elevates T␤RII homodimerization to some degree and strongly enhances T␤RI⅐T␤RII heteromeric complex formation. Both ligand-induced effects depend on the region encompassed between residues 200 -242 of T␤RII. Furthermore, the kinase activity of T␤RI is also necessary for the latter effect. All forms of the homo-and hetero-oligomers, whether constitutively present on the membrane or formed upon TGF-␤1 stimulation, were stable in the time-scale of our patch/FRAP measurements. We suggest that the different forms of receptor oligomerization may serve as a basis for the heterogeneity of TGF-␤ signaling responses. Transforming growth factor-␤ (TGF-␤)3 comprises a large superfamily of cysteine knot growth factors which regulate diverse biological processes including cell proliferation, differentiation, migration, and death (1-4). They were implicated in embryonic development, immune responses, hematopoiesis, and cancer (1, 2, 4 -6). TGF-␤ signals via two receptor Ser/Thr kinases, type I and type II (T␤RI and T␤RII) (3, 4, 7-9). T␤RII can bind ligands but requires T␤RI for signaling, whereas T␤RI alone is incapable of ligand binding (8, 10 -13). T␤RII is a constitutively active kinase regulated by autophosphorylation (14,15). In the presence of ligand, T␤RII phosphorylates specific Ser residues in T␤RI, mediating its activation (13, 16). In turn, T␤RI phosphorylates Smad2/3 proteins, mediating their translocation together with Smad4 to the nucleus, where they regulate transcription of target genes (2-4, 17).T␤RI and T␤RII can physically associate (13, 18 -21). Earlier we demonstrated in live cells that both T␤RI (22) and T␤RII (23) can form homodimers even in the absence of ligand, and biological evidence supports the homo-oligomerization of both receptors (12,15,24,25). On the other hand, the tendency of T␤RI and T␤RII to form heterotetramers is strongly elevated by .Recent studies have show...

Transforming growth factor (TGF)-␤ receptors stimulate diverse signaling processes that control a wide range of biological responses. In polarized epithelia, the TGF␤ type II receptor (T2R) is localized at the basolateral membranes. Sequential cytoplasmic truncations resulted in receptor missorting to apical surfaces, and they indicated an essential targeting element(s) near the receptor's C terminus. Point mutations in the full-length receptor confirmed this prediction, and a unique basolateral-targeting region was elucidated between residues 529 and 538 (LTAxxVAxxR) that was distinct, but colocalized within a clinically significant signaling domain essential for TGF␤-dependent activation of the Smad2/3 cascade. Transfer of a terminal 84 amino-acid fragment, containing the LTAxxVAxxR element, to the apically sorted influenza hemagglutinin (HA) protein was dominant and directed basolateral HA expression. Although delivery to the basolateral surfaces was direct and independent of any detectable transient apical localization, fluorescence recovery after photobleaching demonstrated similar mobility for the wild-type receptor and a missorted mutant lacking the targeting motif. This latter finding excludes the possibility that the domain acts as a cell membrane retention signal, and it supports the hypothesis that T2R sorting occurs from an intracellular compartment. INTRODUCTIONEpithelial cells form highly polarized monolayers that generate two morphologically and functionally distinct domains, an apical luminal facing domain and a basolateral domain that separates the epithelia from the underlying mesenchyme. Because the maintenance of cell polarity is dependent upon the asymmetrical distribution of proteins and lipids to precise locales on the cell surface (Rodriguez-Boulan et al., 2005), a number of cis-acting targeting signals have been described mediating protein sorting. For example, basolateral delivery is often regulated by minimal amino acid motifs in the cytoplasmic region of a wide range of membrane proteins, often being localized to juxtamembrane locales (Aroeti et al., 1998;Ikonen and Simons, 1998; RodriguezBoulan et al., 2005). Although extensive heterogeneity exists, certain features commonly arise in the amino acid sequences. Specifically, the targeting of many basolateral proteins, including the low-density lipoprotein receptor and vesicular stomatitis virus glycoprotein, have been demonstrated to be regulated by tyrosine-based motifs (Matter et al., 1992;Thomas et al., 1993) and a consensus sequence, YXX (where X is any amino acid and represents a large hydrophobic residue), has been proposed to be needed for correct trafficking (Hunziker and Mellman, 1991;Matter et al., 1992;Honing and Hunziker, 1995;Aroeti et al., 1998). Moreover, bipartite sorting signals and dihydrophobic residues such as dileucine (LL) motifs have also figured highly as effectors of basolateral trafficking (Hunziker and Fumey, 1994;Miranda et al., 2001). However, for many other reported basolaterally located proteins, the exac...

Background Activins and bone morphogenetic proteins (BMPs) play critical, sometimes opposing roles, in multiple physiological and pathological processes and diseases. They signal to distinct Smad branches; activins signal mainly to Smad2/3, while BMPs activate mainly Smad1/5/8. This gives rise to the possibility that competition between the different type I receptors through which activin and BMP signal for common type II receptors can provide a mechanism for fine-tuning the cellular response to activin/BMP stimuli. Among the transforming growth factor-β superfamily type II receptors, ACVR2A/B are highly promiscuous, due to their ability to interact with different type I receptors (e.g., ALK4 vs. ALK2/3/6) and with their respective ligands [activin A (ActA) vs. BMP9/2]. However, studies on complex formation between these full-length receptors situated at the plasma membrane, and especially on the potential competition between the different activin and BMP type I receptors for a common activin type II receptor, were lacking. Results We employed a combination of IgG-mediated patching-immobilization of several type I receptors in the absence or presence of ligands with fluorescence recovery after photobleaching (FRAP) measurements on the lateral diffusion of an activin type II receptor, ACVR2A, to demonstrate the principle of competition between type I receptors for ACVR2. Our results show that ACVR2A can form stable heteromeric complexes with ALK4 (an activin type I receptor), as well as with several BMP type I receptors (ALK2/3/6). Of note, ALK4 and the BMP type I receptors competed for binding ACVR2A. To assess the implications of this competition for signaling output, we first validated that in our cell model system (U2OS cells), ACVR2/ALK4 transduce ActA signaling to Smad2/3, while BMP9 signaling to Smad1/5/8 employ ACVR2/ALK2 or ACVR2/ALK3. By combining ligand stimulation with overexpression of a competing type I receptor, we showed that differential complex formation of distinct type I receptors with a common type II receptor balances the signaling to the two Smad branches. Conclusions Different type I receptors that signal to distinct Smad pathways (Smad2/3 vs. Smad1/5/8) compete for binding to common activin type II receptors. This provides a novel mechanism to balance signaling between Smad2/3 and Smad1/5/8.

Both transforming growth factor beta (TGF-␤) and p53 have been shown to control normal cell growth. Acquired mutations either in the TGF-␤ signaling pathway or in the p53 protein were shown to induce malignant transformation. Recently, cross talk between wild-type p53 and the TGF-␤ pathway was observed. The notion that mutant p53 interferes with the wild-type p53-induced pathway and acts by a "gain-of-function" mechanism prompted us to investigate the effect of mutant p53 on the TGF-␤-induced pathway. In this study, we show that cells expressing mutant p53 lost their sensitivity to TGF-␤1, as observed by less cell migration and a reduction in wound healing. We found that mutant p53 attenuates TGF-␤1 signaling. This was exhibited by a reduction in SMAD2/3 phosphorylation and an inhibition of both the formation of SMAD2/SMAD4 complexes and the translocation of SMAD4 to the cell nucleus. Furthermore, we found that mutant p53 attenuates the TGF-␤1-induced transcription activity of SMAD2/3 proteins. In searching for the mechanism that underlies this attenuation, we found that mutant p53 reduces the expression of TGF-␤ receptor type II. These data provide important insights into the molecular mechanisms that underlie mutant p53 "gain of function" pertaining to the TGF-␤ signaling pathway.

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