Functional roles for the cytoplasmic domain of the type III transforming growth factor beta receptor in regulating transforming growth factor beta signaling.

Transforming growth factor beta (TGF-beta) signals through three high affinity cell surface receptors, TGF-beta 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-beta ligand to the type II receptor. The short cytoplasmic domain of the type III receptor is thought to have no role in TGF-beta 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-beta, to bind the type II receptor, and to enhance TGF-beta binding to the type II receptor but is unable to enhance TGF-beta2 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-beta signaling.

Mapping of putative binding sites on the ectodomain of the type II TGF-beta receptor by scanning-deletion mutagenesis and knowledge-based modeling.

Binding surfaces of the type II transforming growth factor (TGF)-beta receptor extracellular domain (TbetaRII-ECD) are mapped by combining scanning-deletion mutagenesis results with knowledge-based modeling of the ectodomain structure. Of the 17 deletion mutants produced within the core binding domain of TbetaRII-ECD, only three retained binding to TGF-beta. Comparative modeling based on the crystal structure of the activin type II receptor extracellular domain (ActRII-ECD) indicates that the TbetaRII mutants which retain TGF-beta binding are deleted in some of the loops connecting the beta-strands in the TbetaRII-ECD model. Interpretation of the mutagenesis data within the structural framework of the ectodomain model allows for the prediction of potential binding sites at the surface of TbetaRII-ECD.

Novel "restoration of function" mutagenesis strategy to identify amino acids of the delta-opioid receptor involved in ligand binding.

A novel "restoration of function" mutagenesis strategy was developed to identify amino acid sequence combinations necessary to restore the ability to bind delta-selective ligands to an inactive delta/mu receptor chimera in which 10 amino acids of the third extracellular loop of the delta receptor were replaced by the corresponding amino acids from the mu receptor (delta/mu291-300). This chimera binds a nonselective opioid ligand but is devoid of affinity for delta-selective ligands. A library of mutants was generated in which some of the 10 amino acids of the mu sequence of delta/mu291-300 were randomly reverted to the corresponding delta amino acid. Using a ligand binding assay, we screened this library to select mutants with high affinity for delta-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 delta-opioid receptor were present in all revertants. Single and double point mutations were then introduced in delta/mu291-300 to evaluate the contribution of the leucine 300 and arginine 291 residues for the binding of delta-selective ligands. An increased affinity for delta-selective ligands was observed when the tryptophan 300 (mu residue) of delta/mu291-300 was reverted to a leucine (delta residue). Further site-directed mutagenesis experiments suggested that the presence of a tryptophan at position 300 may block the access of delta-selective ligands to their docking site.

Scanning-deletion analysis of the extracellular domain of the TGF-beta receptor type II.

There are three main types of receptors for TGF-beta termed receptor type I, type II and type III. TGF-beta receptor type II has a crucial role in the cell's responsiveness to TGF-beta as it is mandatory for the TGF-beta binding to the signaling complex (receptor type I and type II). Here we have used a scanning-deletion mutagenesis approach to determine the core binding domain of the extracellular domain of receptor type II that is required for interaction with TGF-beta. Deletions of three amino acids were systematically introduced at intervals of five amino acids in order to scan the N- and C-terminus of the extracellular domain of the receptor. We find that the N-terminal region which is devoid of cysteine residues is not critical for ligand binding. Similarly, the C-terminal region, i.e., the amino acids flanking the transmembrane domain, are dispensable for binding. These results suggest that the central 100 amino acid span that is rich in cysteine residues is the core binding domain for TGF-beta.

Mutagenesis analysis of the membrane-proximal ligand binding site of the TGF-beta receptor type III extracellular domain.

There are two TGF-beta binding subdomains in the extracellular domain of receptor type III (proximal and distal in relation to the transmembrane domain). Here we present an extension of our analysis of the proximal binding site of receptor type III. Due to the original deletion mutagenesis strategy, our proximal binding site contained 19 amino acids from the N-terminal part of the receptor. By deleting these, we demonstrated that they did not contribute to the binding ability of the proximal binding site. We also produced a soluble, secreted form of the proximal binding site and demonstrated that it was able to bind TGF-beta. Finally, we analyzed the role of the three asparagine residues (580, 591, 595) that are located in the region of the receptor that is necessary for expression of a functional proximal binding site, and found that mutation of these residues individually to alanine did not affect ligand binding.

Mapping of the ligand binding domain of the transforming growth factor beta receptor type III by deletion mutagenesis.

Transforming growth factor beta (TGF-beta) receptor type III is a membrane-anchored proteoglycan that binds TGF-beta via the core protein. We have determined, by deletion mutagenesis of the receptor type III, the minimal essential region of the extracellular domain that is capable of binding TGF-beta. Nine deletion mutants were produced, six of which are expressed on the cell surface and bind TGF-beta. We find that the shortest of these active mutants, which retains only 253 of the 785 amino acids of the extracellular domain, binds TGF-beta with the same affinity as the full-length receptor. These results indicate that the ligand binding domain lies proximal to the transmembrane domain and is functionally independent from the rest of the extracellular domain. We have determined from the mutants that one of the potential glycosaminoglycan attachment sites in the receptor type III is not utilized. Results from the nonglycosylated mutants confirm that the glycosaminoglycan chains are not required for the folding, targeting, and TGF-beta binding activity of the receptor. Moreover, we present evidence for dimerization and multimerization of the receptor.

Energy balance and lipid metabolism in transgenic mice bearing an antisense GCR gene construct.

Energy balance and lipid metabolism were investigated in transgenic mice bearing an antisense glucocorticoid receptor (GCR) gene construct that impairs the normal expression of the GCR gene. Food intake was recorded during the 15 days preceding decapitation of adult normal and transgenic mice, and feces were collected to derive the digestible energy intake. Body composition measurements consisted of the determination of energy, protein, and fat content of the carcass. Carcass energy was determined by bomb calorimetry, whereas carcass protein was measured by the Kjeldahl procedure. Energy expenditure was estimated from the continuous oxygen consumption (VO2) monitoring over a 24-h period. Lipoprotein lipase (LPL) activity was quantified in epididymal white adipose tissue (WAT), heart, and vastus lateralis muscle (VLM) by measuring the in vitro hydrolysis of labeled triolein in the presence of tissue homogenates. Norepinephrine (NE) content of both interscapular brown adipose tissue (BAT) and heart were determined by high-performance liquid chromatography (HPLC). Energy intake and expenditure were significantly lower in transgenic mice than in controls. Concurrently, both fat content and total energy of the carcasses were significantly higher in the transgenic animals. In comparison with normal mice, heart and VLM LPL activity was significantly reduced in transgenic mutants. There was no difference between groups in LPL activity in WAT. Finally, heart and BAT NE contents were lower in transgenic animals than in control mice. These results suggest that a defective GCR system may affect energy balance through increasing energetic efficiency, and they emphasize the modulatory effects of hypothalamic-pituitary-adrenal axis changes on muscle LPL activity.

Antidepressant drug action in a transgenic mouse model of the endocrine changes seen in depression.

We have created transgenic mouse lines with impaired glucocorticoid receptor function by expression of a type II glucocorticoid receptor antisense RNA in brain tissues. These animals have endocrinological characteristics similar to those seen in depression, including a hyperactive hypothalamic-pituitary-adrenal axis as indicated by elevated plasma corticosterone and adrenocorticotropin hormone levels. Treatment of transgenic animals with the tricyclic antidepressant desipramine increased hypothalamic glucocorticoid receptor mRNA concentration and dexamethasone-binding activity while decreasing plasma adrenocorticotropin hormone concentration and corticosterone levels. These results support the hypothesis that antidepressants exert action on the hypothalamic-pituitary-adrenal axis through modulation of glucocorticoid receptor gene expression.

Increased glucocorticoid receptor gene promoter activity after antidepressant treatment.

We have tested the hypothesis that antidepressants affect the expression of the glucocorticoid receptor gene, by looking at glucocorticoid receptor gene promoter activity, glucocorticoid receptor mRNA levels, and glucocorticoid-binding activity after treatment of different cell lines with desipramine. Treatment of LTK- cells or Neuro 2A cells with desipramine produced a 50-200% increase in chloramphenicol acetyltransferase activity transcribed from a 2.7-kilobase glucocorticoid receptor gene promoter region. In cell lines derived from both neuronal and non-neuronal sources, glucocorticoid receptor mRNA concentration doubled after desipramine treatment, and this was associated with a 2-fold higher functional glucocorticoid binding capacity and increased glucocorticoid sensitivity, as measured with the reporter plasmid pMMTVCAT. Antidepressant-induced increases in glucocorticoid receptor gene promoter activity, glucocorticoid receptor mRNA levels, and functional glucocorticoid binding activity suggest a novel mechanism of action for these drugs on the hypothalamic-pituitary-adrenal axis.

Impaired type II glucocorticoid-receptor function in mice bearing antisense RNA transgene.

Glucocorticoids, in conjunction with their cognate receptors, exert negative-feedback effects on the hypothalamus-pituitary-adrenal axis, suppressing adrenal steroid secretions. Two types of corticosteroid receptor, distinguishable by their ability to bind corticosterone, have been identified as classical mineralocorticoid (type I) and glucocorticoid (type II) receptors by cloning their complementary DNAs. The type I receptor controls the basal circadian rhythm of corticosteroid secretion. Both receptor types are involved in negative feedback, but the type II receptor may be more important for terminating the stress response as it is the only one to be increased in animals rendered more sensitive to corticosteroid negative-feedback effects. Here we create a transgenic mouse with impaired corticosteroid-receptor function by partially knocking out gene expression with type II glucocorticoid receptor antisense RNA. We use this animal to study the glucocorticoid feedback effect on the hypothalamus-pituitary-adrenal axis.

Decreased glucocorticoid receptor activity following glucocorticoid receptor antisense RNA gene fragment transfection.

Depression is often characterized by increased cortisol secretion caused by hyperactivity of the hypothalamic-pituitary-adrenal axis and by nonsuppression of cortisol secretion following dexamethasone administration. This hyperactivity of the hypothalamic-pituitary-adrenal axis could result from a reduced glucocorticoid receptor (GR) activity in neurons involved in its control. To investigate the effect of reduced neuronal GR levels, we have blocked cellular GR mRNA processing and/or translation by introduction of a complementary GR antisense RNA strand. Two cell lines were transfected with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under control of the mouse mammary tumor virus long terminal repeat (a glucocorticoid-inducible promoter). This gene construction permitted assay of the sensitivity of the cells to glucocorticoid hormones. Cells were also cotransfected with a plasmid containing 1,815 bp of GR cDNA inserted in the reverse orientation downstream from either a neurofilament gene promoter element or the Rous sarcoma virus promoter element. Northern (RNA) blot analysis demonstrated formation of GR antisense RNA strands. Measurement of the sensitivity of CAT activity to exogeneous dexamethasone showed that although dexamethasone increased CAT activity by as much as 13-fold in control incubations, expression of GR antisense RNA caused a 2- to 4-fold decrease in the CAT response to dexamethasone. Stable transfectants bearing the GR antisense gene fragment construction demonstrated a 50 to 70% decrease of functional GR levels compared with normal cells, as evidenced by a ligand-binding assay with the type II glucocorticoid receptor-specific ligand [3H]RU 28362. These results validate the use of antisense RNA to GR to decrease cellular response to glucocorticoids.

Differential regulation by dexamethasone of glucocorticoid receptor messenger RNA concentrations in neuronal cultures derived from fetal rat hypothalamus and cerebral cortex.

1. Differential regulation, by dexamethasone, of glucocorticoid receptor gene expression was studied in three different neuronal cultures derived from hypothalamus amygdala, and cerebral cortex. 2. Cellular glucocorticoid receptor (GR) mRNA concentration was measured by hybridization using a 32P-labeled RNA probe complementary to a 2.2-kb fragment of the glucocorticoid receptor mRNA. Changes in the amount of GR mRNA were evaluated in relation to the content of beta-actin mRNA. 3. In cells derived from either hypothalamus or cerebral cortex, we observed a complex pattern of GR mRNA concentrations which were characterized by cyclic variations of GR mRNA content during continuous treatment with dexamethasone for up to 72 hr. 4. In contrast to cells derived from the hypothalamus where a persistent 30-40% reduction in GR mRNA levels was seen for up to a least 72 hr, we observed, in cells derived from the cerebral cortex, a sustained increased (1.4-fold) of the GR mRNA at this same time interval.

Antidepressants regulate glucocorticoid receptor messenger RNA concentrations in primary neuronal cultures.

Increased cortisol secretion, caused by hyperactivity of the brain-pituitary-adrenal axis, and non-suppression of cortisol secretion following dexamethasone administration are two characteristics frequently associated with major depression or the depressed phase of bipolar illness. Antidepressants, irrespective of their selective inhibitory actions on the re-uptake of serotonin or of norepinephrine, modify glucocorticoid receptor messenger RNA concentrations in primary cultures of rat hypothalamic or amygdaloid neurons in a biphasic manner, with predominant stimulatory effects. This suggests a mechanism whereby antidepressants, by restoring the sensitivity of the limbic-hypothalamic system to glucocorticoid feedback inhibition, reverse the hyperactivity of the brain-pituitary-adrenal axis.