Herbert Y. Lin

Expression cloning of the TGF-β type II receptor, a functional transmembrane serine/threonine kinase

A cDNA encoding the TGF-beta type II receptor protein has been isolated by an expression cloning strategy. The cloned cDNA, when transfected into COS cells, leads to overexpression of an approximately 80 kd protein that specifically binds radioiodinated TGF-beta 1. Excess TGF-beta 1 competes for binding of radioiodinated TGF-beta 1 in a dose-dependent manner and is more effective than TGF-beta 2. The predicted receptor structure includes a cysteine-rich extracellular domain, a single hydrophobic transmembrane domain, and a predicted cytoplasmic serine/threonine kinase domain. A chimeric protein containing the intracellular domain of the type II receptor and expressed in E. coli can phosphorylate itself on serine and threonine residues in vitro, indicating that the cytoplasmic domain of the type II receptor is a functional kinase. This result implicates serine/threonine phosphorylation as an important mechanism of TGF-beta receptor-mediated signaling.

Expression cloning and characterization of the TGF-β type III receptor

The rat TGF-beta type III receptor cDNA has been cloned by overexpression in COS cells. The encoded receptor is an 853 amino acid protein with a large N-terminal extracellular domain containing at least one site for glycosaminoglycan addition, a single hydrophobic transmembrane domain, and a 41 amino acid cytoplasmic tail with no obvious signaling motif. Introduction of the cDNA into COS cells and L6 myoblasts induces expression of a heterogenously glycosylated 280-330 kd protein characteristic of the type III receptor that binds TGF-beta 1 specifically. In L6 myoblasts lacking the endogenous type III receptor, expression of the recombinant receptor leads to an increase in the amount of ligand bound and cross-linked to surface type II TGF-beta receptors. This indicates that the type III receptor may regulate the ligand-binding ability or surface expression of the type II receptor.

Receptors for the TGF-β superfamily: multiple polypeptides and serine/threonine kinases

Members of the transforming growth factor beta (TGF-beta) superfamily of peptide growth factors have profound effects on the growth and differentiation of many cell types. Insights into the poorly understood mechanisms of action of these ligands have come from the recent molecular cloning of two types of high-affinity receptors - type II and type III - for TGF-beta superfamily members. The cell surface expression of the type III receptor, a membrane-bound proteoglycan, appears to modulate the binding of ligand to the type II receptor, which is a transmembrane serine/threonine kinase. These results provide evidence for interactions between different receptor types, and suggest that serine/threonine phosphorylation is an important element in TGF-beta-induced signalling.

The Soluble Exoplasmic Domain of the Type II Transforming Growth Factor (TGF)-β Receptor A HETEROGENEOUSLY GLYCOSYLATED PROTEIN WITH HIGH AFFINITY AND SELECTIVITY FOR TGF-β LIGANDS

The transforming growth factor (TGF)-β type II receptor is a transmembrane serine/threonine kinase which is essential for all TGF-β-induced signals. In several cell types TGF-β2 is as potent as TGF-β1 or TGF-β3 in inducing cellular responses, yet TGF-β2 does not bind to the majority of expressed type II receptors. Here we characterized the properties of the soluble extracellular domain of the human TGF-β type II receptor synthesized in COS-7 cells. Like the membrane-attached type II receptor, the soluble receptor contains complex N-linked oligosaccharides as well as additional sialic acid residues that cause it to migrate heterogenously upon SDS-polyacrylamide gel electrophoresis. 125I-TGF-β1 binds to and is chemically cross-linked to this protein. Unlabeled TGF-β1 inhibits the binding of 125I-TGF-β1 with an apparent dissociation constant (Kd) of ∼200 pM, similar to the apparent Kd (∼ 50 pM) of the cell-surface type II receptor. TGF-β3 inhibits the binding of 125I-TGF-β1 to the soluble type II receptor with a similar dissociation constant, ∼500 pM. In contrast, 125I-TGF-β2 cannot bind and be chemically cross-linked to the soluble type II receptor, nor does as much as a 125-fold excess of unlabeled TGF-β2 inhibit the binding of 125I-TGF-β1 to the soluble receptor. This is the first demonstration of the binding affinities of the type II receptor in the absence of the other cell-surface molecules known to bind TGF-β. Expressed alone in COS-7 cells the type II receptor also cannot bind TGF-β2; co-expression of type III receptor enables the type II receptor to bind TGF-β2. Thus, the type III receptor or some other component is required for transmission of TGF-β2-induced signals by the type II receptor.

Endothelin-induced endocytosis of cell surface ETA receptors. Endothelin remains intact and bound to the ETA receptor.

We demonstrate unusual features of the intracellular processing of endothelin-1 (ET-1) and its receptor ETA, the receptor subtype that mediates contraction of vascular smooth muscle cells. First, we show that in stably transfected CHO cells expressing ETA, binding of an ET-1 ligand induces rapid endocytosis of cell surface ETA. Receptor endocytosis was measured both by immunofluorescence and by radioiodinated antibodies specific for ETA. Second, we demonstrate that ET-1 remains intact for up to 2 h after endocytosis and, as judged by co-immunoprecipitation, internalized I-ET-1 remains bound to ETA receptors. We hypothesize that internalized ET-1, bound to ETA receptors, continues to activate a signal-transducing G protein, thus accounting for the prolonged period of contraction induced in smooth muscle cells by a single administration of ET-1.

Biosynthesis of the type I and type II TGF-beta receptors. Implications for complex formation.

The TGF-β type I and type II receptors (TβRI and TβRII) are signaling receptors that form heteromeric cell surface complexes with the TGF-βs as one of the earliest events in the cellular response to these multifunctional growth factors. Using TGF-β-responsive mink lung epithelial cells (Mv1Lu), we have determined the half-lives of the endoplasmic reticulum (ER) and mature forms of these receptors. In metabolically labeled cells, approximately 90% of newly synthesized type II receptor undergoes modification of N-linked sugars in the Golgi, with a half-life of 30-35 min; the Golgi-processed form of the receptor has a relatively short metabolic half-life of 2.5 h. In contrast, only 50% of pulse-labeled type I receptor is converted to the Golgi-processed and therefore endoglycosidase H-resistant form, and the endoglycosidase H-sensitive ER form has a half-life of 2.8-3 h. Addition of 100 pM TGF-β1 causes the Golgi-processed type II receptor to become less stable, with a half-life of 1.7 h, and also destabilizes the Golgi-processed type I receptor. TGF-β1 binding and cross-linking experiments on cells treated with tunicamycin for various times confirm different ER to cell surface processing times for TβRI and TβRII. Our results, which suggest that stable complexes between type I and II TGF-β receptors do not form until the proteins reach a post-ER compartment (presumably the cell surface), have important implications for our understanding of complex formation and receptor regulation.

- TGFβRII: TGF-β type II receptor (vertebrates)

The transforming growth factor-β (TGFβ) type II receptor is a transmembrane serine/threonine kinase that binds TGFβ1 and TGFβ3 with greater affinity than TGFβ2. It is thought to mediate the multiple effects of TGFβ, such as increased extracellular matrix production and inhibition of cell growth. The signal transduction pathway and the substrate specificities of this receptor kinase are not known. The kinase domain has four inserts when compared to mouse PKAα. It is unclear what the function of these inserts is, but they are in regions where they may serve to position loops that interact with substrate. It is not known whether dimerization is necessary for kinase function. There are several in vitro autophosphorylation sites. TGFβ type II receptors have been shown to autophosphorylate on serines and threonines. No other substrate has been identified.