Transforming growth factor-beta (TGF-beta) superfamily members regulate a wide range of biological processes by binding to two transmembrane serine/threonine kinase receptors, type I and type II. We have previously shown that the internalization of these receptors is inhibited by K(+) depletion, cytosol acidification, or hypertonic medium, suggesting the involvement of clathrin-coated pits. However, the involvement of the clathrin-associated adaptor complex AP2 and the identity of the AP2 subunit that binds the receptors were not known. Herein, we have studied these issues by combining studies on intact cells with in vitro assays. Using fluorescence photobleaching recovery to measure the lateral mobility of the receptors on live cells (untreated or treated to alter their coated pit structure), we demonstrated that their mobility is restricted by interactions with coated pits. These interactions were transient and mediated through the receptors' cytoplasmic tails. To measure direct binding of the receptors to specific AP2 subunits, we used yeast two-hybrid screens and in vitro biochemical assays. In contrast to most other plasma membrane receptors that bind to AP2 via the mu2 subunit, AP2/TGF-beta receptor binding was mediated by a direct interaction between the beta2-adaptin N-terminal trunk domain and the cytoplasmic tails of the receptors; no binding was observed to the mu2, alpha, or sigma2 subunits of AP2 or to mu1 of AP1. The data uniquely demonstrate both in vivo and in vitro the ability of beta2-adaptin to directly couple TGF-beta receptors to AP2 and to clathrin-coated pits, providing the first in vivo evidence for interactions of a transmembrane receptor with beta2-adaptin.

Transforming growth factor-␤s (TGF-␤) are multifunctional proteins capable of either stimulating or inhibiting mitosis, depending on the cell type. These diverse cellular responses are caused by stimulating a single receptor complex composed of type I and type II receptors. Using a chimeric receptor model where the granulocyte/monocyte colony-stimulating factor receptor ligand binding domains are fused to the transmembrane and cytoplasmic signaling domains of the TGF-␤ type I and II receptors, we wished to describe the role(s) of specific amino acid residues in regulating ligand-mediated endocytosis and signaling in fibroblasts and epithelial cells. Specific point mutations were introduced at Y182, T200, and Y249 of the type I receptor and K277 and P525 of the type II receptor. Mutation of either Y182 or Y249, residues within two putative consensus tyrosine-based internalization motifs, had no effect on endocytosis or signaling. This is in contrast to mutation of T200 to valine, which resulted in ablation of signaling in both cell types, while only abolishing receptor down-regulation in fibroblasts. Moreover, in the absence of ligand, both fibroblasts and epithelial cells constitutively internalize and recycle the TGF-␤ receptor complex back to the plasma membrane. The data indicate fundamental differences between mesenchymal and epithelial cells in endocytic sorting and suggest that ligand binding diverts heteromeric receptors from the default recycling pool to a pathway mediating receptor down-regulation and signaling. INTRODUCTIONTransforming growth factor-␤s (TGF-␤) control a variety of cellular processes as diverse as mitotic inhibition or stimulation (Massagué, 1996;Moses and Serra, 1996). It is unclear how the same receptor complex can mediate such different cellular phenotypes. The most commonly accepted receptor model for TGF-␤ action consists of a heteromeric complex composed of type I and type II receptors (Wrana et al., 1992(Wrana et al., , 1994. Once associated, the type I receptor becomes phosphorylated primarily within the juxtamembrane GS domain (amino acids 185-192) by the constitutive serine/threonine kinase activity of the type II receptor (Franzén et al., 1995;Wieser et al., 1995). Phosphorylation of the GS domain is proposed to activate the type I receptor, resulting in signal propagation to downstream effector molecules (Massagué, 1998). In addition, specific residues in nearby regions have also been suggested to have both positive and negative regulatory functions (Wieser et al., 1995;Charng et al., 1996;Souchelnytskyi et al., 1996;Doré et al., 1998). For instance, threonine 200 has been shown to have a fundamental role in mediating all aspects of TGF-␤ signaling (Wieser et al., 1995), whereas replacement of threonine 204 with an acidic residue, such as aspartate, can generate a type I receptor capable of signaling (albeit to a lesser extent) independent of ligand or an associated type II receptor (Wieser et al., 1995;Charng et al., 1996;Luo and Lodish, 1996). What makes these data most i...

Transforming growth factor beta (TGF-beta) coordinates a number of biological events important in normal and pathophysiological growth. In this study, deletion and substitution mutations were used to identify receptor motifs modulating TGF-beta receptor activity. Initial experiments indicated that a COOH-terminal sequence between amino acids 482-491 in the kinase domain of the type I receptor was required for ligand-induced receptor signaling and down-regulation. These 10 amino acids are highly conserved in mammalian, Xenopus, and Drosophila type I receptors. Although mutation or deletion of the region (referred to as the NANDOR BOX, for nonactivating non-down-regulating) abolishes TGF-beta-dependent mitogenesis, transcriptional activity, type I receptor phosphorylation, and down-regulation in mesenchymal cultures, adjacent mutations also within the kinase domain are without effect. Moreover, a kinase-defective type I receptor can functionally complement a mutant BOX expressing type I receptor, documenting that when the BOX mutant is activated, it has kinase activity. These results indicate that the sequence between 482 and 491 in the type I receptor provides a critical function regulating activation of the TGF-beta receptor complex.

Transforming growth factor-␤ (TGF-␤) family polypeptides regulate cell growth and differentiation by binding to single pass serine/threonine kinases referred to as TGF-␤ type I and II receptors. Although interaction screens have shown that the immunophilin FKBP12 interacts with TGF-␤ type I receptors, the role of FKBP12 in TGF-␤ receptor action is presently unclear. Using a chimeric TGF-␤ receptor system, we have shown a specific enhancement of internalization when FKBP12 binding to the type I receptor was prevented with rapamycin. Moreover, although earlier studies demonstrated that type II receptor kinase activity was required for optimal internalization in mesenchymal cells, we found that rapamycin functioned downstream of the type II receptor kinase. Thus, rather than modulating TGF-␤ signaling, our data suggest a novel role for FKBP12 as a negative regulator of TGF-␤ receptor endocytosis.The transforming growth factor-␤ (TGF-␤) 1 superfamily regulates a wide range of biological processes (1-3). A variety of structural and functional criteria have been used to group the superfamily into three classes consisting of TGFs-␤, activins, and bone morphogenic proteins. Members of these groups have been implicated in the regulation of wound healing, cellular proliferation, immune responses, and pattern formation throughout development.Two distinct membrane receptor proteins referred to as TGF-␤ type I (TGF-␤RI) and type II (TGF-␤RII) receptors and a membrane-anchored proteoglycan called the type III receptor have been characterized and show high affinity TGF-␤ ligand binding (4 -6). The type III receptor is proposed to present TGF-␤ to the signaling receptors and to enhance cell responsiveness to TGF-␤; however, its short intracellular domain and absence of known signaling motifs suggest a limited role in direct regulation of TGF-␤ signaling. TGF-␤ signaling is primarily mediated by TGF-␤RI and TGF-␤RII. The signaling cascade begins when TGF-␤RII binds ligand and recruits TGF-␤RI into a heteromeric complex. TGF-␤RII then transphosphorylates TGF-␤RI at serine residues in the juxtamembrane glycine/serine-rich domain, and the activated TGF-␤RI, in turn, interacts and activates downstream targets (7).Interaction screens have demonstrated that the immunophilin FKBP12 binds to TGF-␤ type I receptors (8 -10). FKBP12 belongs to a large family of peptidylprolyl cis-trans isomerases catalyzing the isomerization of peptidylprolyl imide bonds in peptide and protein substrates. It was first isolated as the receptor protein for the immunosuppressive drugs FK506 and rapamycin (11,12). The FKBP12⅐FK506 complex inhibits calcineurin phosphatase activity and nuclear factor of activated T cells translocation, whereas the FKBP12⅐rapamycin complex inactivates TOR (target of rapamycin) family proteins (13,14). Although the interaction of FKBP12 and the TGF-␤ type I receptor has been unequivocally established, its function in TGF-␤-mediated signaling has been controversial. Initial reports proposed that FKBP12 functioned as a negative regu...