Differential Requirement for Type I and Type II Transforming Growth Factor β Receptor Kinase Activity in Ligand-mediated Receptor Endocytosis

Abstract

Transforming growth factor ␤ (TGF␤) superfamily polypeptides regulate cell growth and differentiation by binding to single pass serine/threonine kinases referred to as TGF␤ type I and type II receptors. Signal propagation is dependent upon heteromeric (type I-type II) complex formation and transphosphorylation of the type I receptor by the type II receptor. While many of the phosphorylation events necessary for receptor signaling have recently been characterized, the role of TGF␤ receptor kinase activity in modulating receptor endocytosis has not been addressed. To that end, we have used chimeric receptors consisting of the extracellular domain of the granulocyte/macrophage colony-stimulating factor ␣ and ␤ receptors spliced to the TGF␤ type I and type II transmembrane and cytoplasmic domains to address the specific role of type I and/or type II receptor kinase activity in TGF␤ receptor internalization, downregulation, and signaling. To inactivate chimeric receptor kinase activity, point mutations in the ATP binding site were made at amino acids 232 and 277 in the type I and type II receptor, respectively. Either of these mutations abolished plasminogen activator inhibitor 1 protein expression stimulated by granulocyte/macrophage colony-stimulating factor activation of chimeric heteromeric type I-type II TGF␤ receptors. They did not, however, modulate TGF␤ signaling stimulated through the endogenous TGF␤ receptor. Although TGF␤ receptor signaling was dependent upon the kinase activity of both chimeric receptors, the initial endocytic response was distinctly regulated by type I and/or type II receptor kinase activity. For instance, while heteromeric receptor complexes containing a kinase-inactive type I receptor were endocytosed similarly to wild type complexes, the kinase activity of the type II TGF␤ receptor was necessary for optimal internalization and receptor down-regulation. Furthermore, these responses were shown to occur independently of type II receptor autophosphorylation but require a type II receptor capable of transphosphorylation.The transforming growth factor ␤ (TGF␤) 1 superfamily of proteins regulate a number of diverse biologic processes (1-3). While the cellular response can be as distinct as growth stimulation or growth inhibition, it appears as though a similar receptor system is utilized for both pathways. Understanding how the receptors are regulated for one family of proteins will ultimately extend the knowledge for the entire superfamily. The model most commonly accepted for receptor activation requires oligomerization of a type I and type II TGF␤ receptor (4 -7). This occurs through ligand binding to a type II receptor and recruitment of a type I receptor into a dimeric and/or tetrameric complex (7-11). The serine/threonine kinase activity of the type I receptor is then activated by specific type II receptor phosphorylations in the juxtamembrane region of the type I receptor (12-16). This cascade of receptor interactions and phosphorylations ultimately results in the propagation of the T...

Endocytic regulation of TGF-β signaling

“…Contrary to what is observed in fibroblasts [46], the kinase activity of TbRII is not essential for downregulation of heteromeric TGF-b receptor complexes in epithelial cells [56]. In addition to this, a signaling-incompetent mutation of TbRI -T200V -abolishes endocytosis/ receptor downregulation in fibroblasts, whereas it has no such effect in epithelial cells [56].…”

“…To overcome this problem and to examine the role of the receptor kinase activity in modulating receptor endocytosis, Leof and colleagues have cleverly engineered artificial receptor constructs by fusing the extracellular domains of the granulocyte/macrophage colony-stimulating factor (GM-CSF) a and b receptors to the transmembrane and cytoplasmic domains of TGF-b type I and type II receptors and followed internalization of the GM-CSF ligand [44]. Using this system, they showed that both TGF-b type I and type II receptors are rapidly endocytosed [45], and that the kinase activity of TbRII, but not TbRI, is required for optimal internalization of the heteromeric receptor complexes [46]. Consistent with this, deletion of the kinase domain of TbRI had no effect on downregulation of the heteromeric receptor complexes [47].…”

“…Interestingly, this motif seems to be essential for TbRI activation as its deletion abolished TbRI phosphorylation by TbRII and the ability of TbRI to mediate TGF-b signaling [47]. As the kinase activity of TbRI may not be necessary for receptor internalization [46], the function of this motif in coupling receptor activation and internalization/downregulation needs further investigation. Supporting the importance of the COOHterminus of type I receptors in mediating receptor endocytosis, a study with the type I activin receptor ALK4 showed that a conserved Trp, six amino acids upstream of the NANDOR Box (W477 in ALK4), is essential for ALK4 internalization, and substitution of this Trp with Ala blocked ligand-induced endocytosis of ALK4 [61].…”

A Novel Transforming Growth Factor-β Receptor-interacting Protein That Is Also a Light Chain of the Motor Protein Dynein

“…In the case of TGF␤, however, the receptor location for either initiation or transmission of TGF␤ signaling activities has not been clearly defined. It has been shown that heteromeric TGF␤ receptors are internalized and down-regulated after TGF␤ activation via a clathrin-dependent mechanism (Anders et al, 1997;Doré et al, 1998) and that the kinase activity of RII is required for these processes to occur optimally (Anders et al, 1998). A more recent report has indicated that Smad phosphorylation does not occur until the GTPase dynamin 2ab excises the budded vesicle from the plasma membrane to form an endocytic vesicle (Penheiter et al, 2002).…”

A Unique Element in the Cytoplasmic Tail of the Type II Transforming Growth Factor-β Receptor Controls Basolateral Delivery

“…The chimeric receptors consist of the ligand binding domains of GM-CSF ␣ or ␤ receptors (Gearing et al, 1989;Hayashida et al, 1990) fused to the transmembrane and cytoplasmic domains of the type I and type II TGF␤ receptors, termed ␣1 and ␤II, respectively (Anders and Leof, 1996;Anders et al, 1997Anders et al, , 1998. Because high-affinity GM-CSF binding and subsequent signaling occurs through the formation of ␣/␤ heterodimers (in a manner analogous to the endogenous TGF␤ receptors), and signals required for basolateral localization have, to date, been solely localized to the cytoplasmic domains in all basolateral proteins studied, the chimeras would be expected to contain all the endogenous signals necessary for basolateral localization.…”