Transforming growth factor ␤ (TGF-␤) signals through three high affinity cell surface receptors, TGF-␤ type I, type II, and type III receptors. The type III receptor, also known as betaglycan, binds to the type II receptor and is thought to act solely by "presenting" the TGF-␤ ligand to the type II receptor. The short cytoplasmic domain of the type III receptor is thought to have no role in TGF-␤ signaling because deletion of this domain has no effect on association with the type II receptor, or with the presentation role of the type III receptor. Here we demonstrate that the cytoplasmic domains of the type III and type II receptors interact specifically in a manner dependent on the kinase activity of the type II receptor and the ability of the type II receptor to autophosphorylate. This interaction results in the phosphorylation of the cytoplasmic domain of the type III receptor by the type II receptor. The type III receptor with the cytoplasmic domain deleted is able to bind TGF-␤, to bind the type II receptor, and to enhance TGF-␤ binding to the type II receptor but is unable to enhance TGF-␤2 signaling, determining that the cytoplasmic domain is essential for some functions of the type III receptor. The type III receptor functions by selectively binding the autophosphorylated type II receptor via its cytoplasmic domain, thus promoting the preferential formation of a complex between the autophosphorylated type II receptor and the type I receptor and then dissociating from this active signaling complex. These studies, for the first time, elucidate important functional roles of the cytoplasmic domain of the type III receptor and demonstrate that these roles are essential for regulating TGF-␤ signaling. Transforming growth factor ␤ (TGF-␤)1 is a member of a family of dimeric polypeptide growth factors which, in addition to the TGF-␤ ligands, includes the bone morphogenetic proteins (BMPs) and the activins (1). TGF-␤ regulates cellular proliferation and differentiation as well as the processes of embryonic development, wound healing, and angiogenesis in a tissue-specific manner. Mutations in TGF-␤ receptors or their intracellular signaling molecules have been described, particularly in association with the development of cancer and hereditary hemorrhagic telangiectasia. Alterations in the production of TGF-␤ ligand have also been linked to numerous disease states, including osteoporosis, hypertension, atherosclerosis, and fibrotic disease of the kidney, liver, and lung (2). TGF-␤ regulates cellular processes by binding to three high affinity cell surface receptors, the TGF-␤ type I, type II, and type III receptors. Where expressed, the type III receptor, also known as betaglycan, is the most abundant TGF-␤ receptor and is traditionally thought to function by binding TGF-␤ and then transferring it to its signaling receptor, the type II receptor. This is particularly important for the TGF-␤2 isoform, which cannot bind the type II receptor independently. The type I and II receptors contain serine/threonine protein k...

A novel "restoration of function" mutagenesis strategy was developed to identify amino acid sequence combinations necessary to restore the ability to bind ␦-selective ligands to an inactive ␦/ receptor chimera in which 10 amino acids of the third extracellular loop of the ␦ receptor were replaced by the corresponding amino acids from the receptor (␦/ 291-300). This chimera binds a nonselective opioid ligand but is devoid of affinity for ␦-selective ligands. A library of mutants was generated in which some of the 10 amino acids of the sequence of ␦/ 291-300 were randomly reverted to the corresponding ␦ amino acid. Using a ligand binding assay, we screened this library to select mutants with high affinity for ␦-selective ligands. Sequence analysis of these revertants revealed that a leucine at position 300, a hydrophobic region (amino acids 295-300), and an arginine at position 291 of the human ␦-opioid receptor were present in all revertants. Single and double point mutations were then introduced in ␦/ 291-300 to evaluate the contribution of the leucine 300 and arginine 291 residues for the binding of ␦-selective ligands. An increased affinity for ␦-selective ligands was observed when the tryptophan 300 ( residue) of ␦/ 291-300 was reverted to a leucine (␦ residue). Further site-directed mutagenesis experiments suggested that the presence of a tryptophan at position 300 may block the access of ␦-selective ligands to their docking site.The opioid receptors are widely distributed throughout the central nervous system and mediate the diverse effects of endogenous opioid peptides and opiate drugs (1). Pharmacological studies have defined at least three classes of opioid receptors, named ␦, , and , that differ in their affinity for ligands and in their distribution in the nervous system (1-6).The antinociception mediated by supraspinal opioid receptors is thought to act via the -opioid receptor subtype (7-10). The numerous side effects accompanying opioid treatment, including respiratory depression and addiction (11), are generally thought to be mediated by the stimulation of receptors. However, there is growing evidence suggesting that selective stimulation of the ␦ receptor may also mediate antinociception (12-19). The strongest indication of an involvement of ␦-opioid receptors in supraspinal antinociception follows from studies with selective antagonists (14 -16). These studies demonstrated that the antinociception produced by intracerebroventricular injection of morphine and [D-Ala 2 ,MePhe 4 ,Gly-ol 5 ]-enkephalin ( agonists) was antagonized by ␤-funaltrexamine and naloxonazine ( antagonists) but not by ICI-174864 (␦ antagonists). Conversely, the antinociception produced by intracerebroventricular injection of DPDPE 1 (␦ agonist) was antagonized by ICI-174864 but not by ␤-funaltrexamine and naloxonazine. Moreover, studies have shown that an antisense oligodeoxynucleotide to the cloned ␦-opioid receptor given intrathecally lowers ␦ but not or spinal (20) and central (21) analgesia. These studies confirm, at the m...