Transforming growth factor beta (TGF beta) family ligands initiate a cascade of events capable of modulating cellular growth and differentiation. The receptors responsible for transducing these cellular signals are referred to as the type I and type II TGF beta receptors. Ligand binding to the type II receptor results in the transphosphorylation and activation of the type I receptor. This heteromeric complex then propagates the signal(s) to downstream effectors. There is presently little data concerning the fate of TGF beta receptors after ligand binding, with conflicting reports indicating no change or decreasing cell surface receptor numbers. To address the fate of ligand-activated receptors, we have used our previously characterized chimeric receptors consisting of the ligand binding domain from the granulocyte/macrophage colony-stimulating factor alpha or beta receptor fused to the transmembrane and cytoplasmic domain of the type I or type II TGF beta receptor. This system not only provides the necessary sensitivity and specificity to address these types of questions but also permits the differentiation of endocytic responses to either homomeric or heteromeric intracellular TGF beta receptor oligomerization. Data are presented that show, within minutes of ligand binding, chimeric TGF beta receptors are internalized. However, although all the chimeric receptor combinations show similar internalization rates, receptor down-regulation occurs only after activation of heteromeric TGF beta receptors. These results indicate that effective receptor down-regulation requires cross-talk between the type I and type II TGF beta receptors and that TGF beta receptor heteromers and homomers show distinct trafficking behavior.

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...