Müllerian Inhibiting Substance lowers testosterone in luteinizing hormone-stimulated rodents

Müllerian Inhibiting Substance (MIS) expression is inversely proportional to the serum concentration of testosterone in males after birth and in vitro studies have shown that MIS can lower testosterone production by Leydig cells. Also, mice overexpressing MIS exhibited Leydig cell hypoplasia and lower levels of serum testosterone, but it is not clear whether this is a result of MIS affecting the development of Leydig cells or their capacity to produce testosterone. To examine the hypothesis that MIS treatment will result in decreased testosterone production by mature Leydig cells in vivo, we treated luteinizing hormone (LH)-stimulated adult male rats and mice with MIS and demonstrated that it can lead to a several-fold reduction in testosterone in serum and in testicular extracts. There was also a slight decrease in 17-OH-progesterone compared to the more significant decrease in testosterone, suggesting that MIS might be regulating the lyase activity of cytochrome P450c17 hydroxylase/lyase (Cyp17), but not its hydroxylase activity. Northern analysis showed that, in both MIS-treated rats and mice, the mRNA for Cyp17, which catalyzes the committed step in androgen synthesis, was down-regulated. In rats, the mRNA for cytochrome P450 side-chain cleavage (P450scc) was also down-regulated by MIS. This was not observed in mice, indicating that there might be species-specific regulation by MIS of the enzymes involved in the testosterone biosynthetic pathway. Our results show that MIS can be used in vivo to lower testosterone production by mature rodent Leydig cells and suggest that MIS-mediated down-regulation of the expression of Cyp17, and perhaps P450scc, contributes to that effect.

An unliganded thyroid hormone receptor causes severe neurological dysfunction

Congenital hypothyroidism and the thyroid hormone (T3) resistance syndrome are associated with severe central nervous system (CNS) dysfunction. Because thyroid hormones are thought to act principally by binding to their nuclear receptors (TRs), it is unexplained why TR knock-out animals are reported to have normal CNS structure and function. To investigate this discrepancy further, a T3 binding mutation was introduced into the mouse TR-β locus by homologous recombination. Because of this T3 binding defect, the mutant TR constitutively interacts with corepressor proteins and mimics the hypothyroid state, regardless of the circulating thyroid hormone concentrations. Severe abnormalities in cerebellar development and function and abnormal hippocampal gene expression and learning were found. These findings demonstrate the specific and deleterious action of unliganded TR in the brain and suggest the importance of corepressors bound to TR in the pathogenesis of hypothyroidism.

Nuclear hormone receptor CHR3 is a critical regulator of all four larval molts of the nematode Caenorhabditis elegans

CHR3 (nhr-23, NF1F4), the homologue of Drosophila DHR3 and mammalian ROR/RZR/RevErbA nuclear hormone receptors, is important for proper epidermal development and molting in the nematode Caenorhabditis elegans. Disruption of CHR3 (nhr-23) function leads to developmental changes, including incomplete molting and a short, fat (dumpy) phenotype. Here, we studied the role of CHR3 during larval development by using expression assays and RNA-mediated interference. We show that the levels of expression of CHR3 (nhr-23) cycle during larval development and reduction of CHR3 function during each intermolt period result in defects at all subsequent molts. Assaying candidate gene expression in populations of animals treated with CHR3 (nhr-23) RNA-mediated interference has identified dpy-7 as a potential gene acting downstream of CHR3. These results define CHR3 as a critical regulator of all C. elegans molts and begin to define the molecular pathway for its function.

Identification and characterization of a second melanin-concentrating hormone receptor, MCH-2R

Melanin-concentrating hormone (MCH) is a 19-aa cyclic neuropeptide originally isolated from chum salmon pituitaries. Besides its effects on the aggregation of melanophores in fish several lines of evidence suggest that in mammals MCH functions as a regulator of energy homeostasis. Recently, several groups reported the identification of an orphan G protein-coupled receptor as a receptor for MCH (MCH-1R). We hereby report the identification of a second human MCH receptor termed MCH-2R, which shares about 38% amino acid identity with MCH-1R. MCH-2R displayed high-affinity MCH binding, resulting in inositol phosphate turnover and release of intracellular calcium in mammalian cells. In contrast to MCH-1R, MCH-2R signaling is not sensitive to pertussis toxin and MCH-2R cannot reduce forskolin-stimulated cAMP production, suggesting an exclusive Gαq coupling of the MCH-2R in cell-based systems. Northern blot and in situ hybridization analysis of human and monkey tissue shows that expression of MCH-2R mRNA is restricted to several regions of the brain, including the arcuate nucleus and the ventral medial hypothalamus, areas implicated in regulation of body weight. In addition, the human MCH-2R gene was mapped to the long arm of chromosome 6 at band 6q16.2–16.3, a region reported to be associated with cytogenetic abnormalities of obese patients. The characterization of a second mammalian G protein-coupled receptor for MCH potentially indicates that the control of energy homeostasis in mammals by the MCH neuropeptide system may be more complex than initially anticipated.

Identification and characterization of a melanin-concentrating hormone receptor

Melanin-concentrating hormone (MCH), a neuropeptide expressed in central and peripheral nervous systems, plays an important role in the control of feeding behaviors and energy metabolism. An orphan G protein-coupled receptor (SLC-1/GPR24) has recently been identified as a receptor for MCH (MCHR1). We report here the identification and characterization of a G protein-coupled receptor as the MCH receptor subtype 2 (MCHR2). MCHR2 has higher protein sequence homology to MCHR1 than any other G protein-coupled receptor. The expression of MCHR2 has been detected in many regions of the brain. In contrast to MCHR1, which is intronless in the coding region and is located at the chromosomal locus 22q13.3, the MCHR2 gene has multiple exons and is mapped to locus 6q21. MCHR2 is specifically activated by nanomolar concentrations of MCH, binds to MCH with high affinity, and signals through Gq protein. This discovery is important for a full understanding of MCH biology and the development of potential therapeutics for diseases involving MCH, including obesity.

Alternative splicing of exon 3 of the human growth hormone receptor is the result of an unusual genetic polymorphism.

Two isoforms of the human growth hormone receptor (hGHR), which differ in the presence (hGHRwt) or absence (hGHRd3) of exon 3, are expressed in the placenta. Specifically, three expression patterns are observed: only hGHRwt, only hGHRd3, or an approximately 1:1 combination of both isoforms. We investigated several potential regulatory mechanisms which might account for the expression of the hGHR isoforms. The frequency of hGHRd3 expression did not change when placentas from differing stages of gestation were examined, suggesting splicing was not developmentally regulated. However, when hGHR isoform expression patterns were examined in each component of a given placenta, it was evident that alternative splicing of exon 3 is individual-specific. Surprisingly, the individual-specific regulation of hGHR isoforms appears to be the result of a polymorphism in the hGHR gene. We analyzed hGHRwt and hGHRd3 expression in Hutterite pedigrees, and our results are consistent with a simple Mendelian inheritance of two differing alleles in which exon 3 is spliced in an "all-or-none" fashion. We conclude the alternative splicing of exon 3 in hGHR transcripts is the result of an unusual polymorphism which significantly alters splicing of the hGHR transcript and that the relatively high frequency (approximately 10%) of homozygous hGHRd3 expression suggests the possibility it may play a role in polygenic determined events.

The nuclear hormone receptor coactivator SRC-1 is a specific target of p300.

p300 and its family member, CREB-binding protein (CBP), function as key transcriptional coactivators by virtue of their interaction with the activated forms of certain transcription factors. In a search for additional cellular targets of p300/CBP, a protein-protein cloning strategy, surprisingly identified SRC-1, a coactivator involved in nuclear hormone receptor transcriptional activity, as a p300/CBP interactive protein. p300 and SRC-1 interact, specifically, in vitro and they also form complexes in vivo. Moreover, we show that SRC-1 encodes a new member of the basic helix-loop-helix-PAS domain family and that it physically interacts with the retinoic acid receptor in response to hormone binding. Together, these results implicate p300 as a component of the retinoic acid signaling pathway, operating, in part, through specific interaction with a nuclear hormone receptor coactivator, SRC-1.

Analysis of estrogen receptor transcriptional enhancement by a nuclear hormone receptor coactivator.

The estrogen receptor (ER), a member of a large superfamily of nuclear hormone receptors, is a ligand-inducible transcription factor that regulates the expression of estrogen-responsive genes. The ER, in common with other members of this superfamily, contains two transcription activation functions (AFs)--one located in the amino-terminal region (AF-1) and the second located in the carboxyl-terminal region (AF-2). In most cell contexts, the synergistic activity of AF-1 and AF-2 is required for full estradiol (E2)-stimulated activity. We have previously shown that a ligand-dependent interaction between the two AF-containing regions of ER was promoted by E2 and the antiestrogen trans-hydroxytamoxifen (TOT). This interaction, however, was transcriptionally productive only in the presence of E2. To explore a possible role of steroid receptor coactivators in transcriptional synergism between AF-1 and AF-2, we expressed the amino terminal (AF-1-containing) and carboxyl-terminal (AF-2-containing) regions of ER as separate polypeptides in mammalian cells, along with the steroid receptor coactivator-1 protein (SRC-1). We demonstrate that SRC-1, which has been shown to significantly increase ER transcriptional activity, enhanced the interaction, mediated by either E2 or TOT, between the AF-1-containing and AF-2-containing regions of the ER. However, this enhanced interaction resulted in increased transcriptional effectiveness only with E2 and not with TOT, consistent with the effects of SRC-1 on the full-length receptor. Our results suggest that after ligand binding, SRC-1 may act, in part, as an adapter protein that promotes the integration of amino- and carboxyl-terminal receptor functions, allowing for full receptor activation. Potentially, SRC-1 may be capable of enhancing the transcriptional activity of related nuclear receptor superfamily members by facilitating the productive association of the two AF-containing regions in these receptors.

Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex.

Transcriptional regulation by nuclear hormone receptors is thought to involve interactions with putative cofactors that may potentiate receptor function. Here we show that human thyroid hormone receptor alpha purified from HeLa cells grown in the presence of thyroid hormone (T3) is associated with a group of distinct nuclear proteins termed thyroid hormone receptor-associated proteins (TRAPs). In an in vitro system reconstituted with general initiation factors and cofactors (and in the absence of added T3), the "liganded" thyroid hormone receptor (TR)/TRAP complex markedly activates transcription from a promoter template containing T3-response elements. Moreover, whereas the retinoid X receptor is not detected in the TR/TRAP complex, its presence is required for the function of the complex. In contrast, human thyroid hormone receptor alpha purified from cells grown in the absence of T3 lacks the TRAPs and effects only a low level of activation that is dependent on added ligand. These findings demonstrate the ligand-dependent in vivo formation of a transcriptionally active TR-multisubunit protein complex and suggest a role for TRAPs as positive coactivators for gene-specific transcriptional activation.

Cytotoxic analogs of luteinizing hormone-releasing hormone containing doxorubicin or 2-pyrrolinodoxorubicin, a derivative 500-1000 times more potent.

Doxorubicin (DOX) and its daunosamine-modified derivative, 2-pyrrolino-DOX, which is 500-1000 times more active than DOX, were incorporated into agonistic and antagonistic analogs of luteinizing hormone-releasing hormone (LH-RH). The conjugation of DOX with LH-RH analogs was performed by using N-(9-fluorenylmethoxycarbonyl)-DOX-14-O-hemiglutarate, a dicarboxylic acid ester derivative of DOX. Coupling this derivative covalently to the epsilon-amino group of the D-Lys side chain of agonist [D-Lys6]LH-RH or antagonistic analog AC-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys-Leu-Arg-Pro-D-Ala-NH 2 [where Nal(2) = 3-(2-naphthyl)alanine, Pal(3) = 3-(3-pyridyl)alanine, and Phe(4CI) = 4-chlorophenylalanine] was followed by the removal of the 9-fluorenylmethoxycarbonyl protective group to yield cytotoxic derivatives of LH-RH analogs containing DOX. From these DOX containing LH-RH hybrids, intensely potent analogs with daunosamine-modified derivatives of DOX can be readily formed. Thus, cytotoxic LH-RH agonist containing DOX (AN-152) can be converted in a 66% yield by a reaction with a 30-fold excess of 4-iodobutyraldehyde in N,N-dimethylformamide into a derivative having 2-pyrrolino-DOX (AN-207). Hybrid molecules AN-152 and AN-207 fully preserve the cytotoxic activity of their radicals, DOX or 2-pyrrolino-DOX, respectively, in vitro, and also retain the high binding affinity of the peptide hormone portion of the conjugates to rat pituitary receptors for LH-RH. These highly potent cytotoxic analogs of LH-RH were designed as targeted anti-cancer agents for the treatment of various tumors that possess receptors for the carrier peptide. Initial in vivo studies show that the hybrid molecules are much less toxic than the respective cytotoxic radicals incorporated and significantly more active in inhibiting tumor growth.

G protein subunits and the stimulation of phospholipase C by Gs-and Gi-coupled receptors: Lack of receptor selectivity of Galpha(16) and evidence for a synergic interaction between Gbeta gamma and the alpha subunit of a receptor activated G protein.

Stimulatory guanine nucleotide binding protein (Gs)-coupled receptors activated by luteinizing hormone, vasopressin, and the catecholamine isoproterenol (luteinizing hormone receptor, type 2 vasopressin receptor, and types 1 and 2 beta-adrenergic receptors) and the Gi-coupled M2 muscarinic receptor (M2R) were expressed transiently in COS cells, alone and in combination with Gbeta gamma dimers, their corresponding Galphas (Galpha(s), or Galpha(i3)) and either Galpha(q) or Galpha(16). Phospholipase C (PLC) activity, assessed by inositol phosphate production from preincorporated myo[3H]inositol, was then determined to gain insight into differential coupling preferences among receptors and G proteins. The following were observed: (i) All receptors tested were able to stimulate PLC activity in response to agonist occupation. The effect of the M2R was pertussis toxin sensitive. (ii) While, as expected, expression of Galpha(q) facilitated an agonist-induced activation of PLC that varied widely from receptor to receptor (400% with type 2 vasopressin receptor and only 30% with M2R), expression of Galpha(16) facilitated about equally well the activation of PLC by any of the tested receptors and thus showed little if any discrimination for one receptor over another. (iii) Gbeta gamma elevated basal (agonist independent) PLC activity between 2- and 4-fold, confirming the proven ability of Gbeta gamma to stimulate PLCbeta. (iv) Activation of expressed receptors by their respective ligands in cells coexpressing excess Gbeta gamma elicited agonist stimulated PLC activities, which, in the case of the M2R, was not blocked by pertussis toxin (PTX), suggesting mediation by a PTX-insensitive PLC-stimulating Galpha subunit, presumably, but not necessarily, of the Gq family. (v) The effects of Gbeta gamma and the PTX-insensitive Galpha elicited by M2R were synergistic, suggesting the possibility that one or more forms of PLC are under conditional or dual regulation of G protein subunits such that stimulation by one sensitizes to the stimulation by the other.

Dominant-negative mutant thyroid hormone receptors prevent transcription from Xenopus thyroid hormone receptor beta gene promoter in response to thyroid hormone in Xenopus tadpoles in vivo.

We describe a dominant-negative approach in vivo to assess the strong, early upregulation of thyroid hormone receptor beta (TR beta) gene in response to thyroid hormone, characteristic of the onset of natural and thyroid hormone-induced amphibian metamorphosis, 3,3',5-Triiodo-thyronine (T3) treatment of organ cultures of premetamorphic Xenopus tadpole tails coinjected in vivo with the wild-type Xenopus TR beta (wt-xTR beta) and three different thyroid responsive element chloramphenicol acetyltransferase (TRE-CAT) reporter constructs, including a direct repeat +4 (DR +4) element in the -200/+87 fragment of the xTR beta promoter, resulted in a 4- to 8-fold enhancement of CAT activity. Two human C-terminal TR beta 1 mutants (delta-hTR beta 1 and Ts-hTR beta 1), an artificial Xenopus C-terminal deletion mutant (mt-xTR beta), and the oncogenic viral homology v-erbA, none of which binds T3, inhibited this T3 response of the endogenous wt-xTR in Xenopus XTC-2 cells cotransfected with the -1600/+87 xTR beta promoter-CAT construct, the potency of the dominant-negative effect of these mutant TRs being a function of the strength of their heterodimerization with Xenopus retinoid X receptor gamma. Coinjection of the dominant-negative Xenopus and human mutant TR beta s into Xenopus tadpole tails totally abolished the T3 responsiveness of the wt-xTR beta with different TREs, including the natural DR +4 TRE of the xTR beta promoter.

Binding in the growth hormone receptor complex.

Binding reactions between human growth hormone (hGH) and its receptor provide a detailed account of how a polypeptide hormone activates its receptor and more generally how proteins interact. Through high-resolution structural and functional studies it is seen that hGH uses two different sites (site 1 and site 2) to bind two identical receptor molecules. This sequential dimerization reaction activates the receptor, presumably by bringing the intracellular domains into close proximity so they may activate cytosolic components. As a consequence of this mechanism it is possible to build antagonists to the receptor by introducing mutations in hGH that block binding at site 2 and to build even more potent antagonists by combining these with mutants that enhance binding at site 1. Alanine-scanning mutagenesis of all contact residues at the site 1 interface shows that only a small and complementary set of side chains clustered near the center of the interface affects binding. The most important contacts are hydrophobic, and these are surrounded by polar and charged interactions of lesser importance. Kinetic analysis shows for the most part that the important side chains function to maintain the complex, not to guide the hormone to the receptor. Hormone-induced homodimerization or heterodimerization reactions are turning out to be pervasive mechanisms for signal transduction. Moreover, the molecular recognition principles seen in the hGH-receptor complex are likely to generalize to other protein-protein complexes.

A protein that interacts with members of the nuclear hormone receptor family: identification and cDNA cloning.

In search of proteins which interact with activated steroid hormone receptors, we screened a human liver lambda gt11 expression library with the glucocorticoid receptor. We identified and cloned a cDNA sequence of 1322 bp that encodes a protein of 274 aa. This protein consists predominantly of hydrophilic amino acids and contains a putative bipartite nuclear localization signal. The in vitro translated receptor-associating protein runs in SDS/polyacrylamide gels with an apparent molecular mass of 46 kDa. By use of the bacterially expressed fusion protein with glutathione S-transferase we have found that interaction is not limited to the glucocorticoid receptor but included other nuclear receptors--most notably, the estrogen and thyroid receptors. Binding also occurs with the glucocorticoid receptor complexed with the antiglucocorticoid RU 38486, with the estrogen receptor complexed with the antiestrogen 4-hydroxytamoxifen or ICI 164,384, and even with receptors not complexed with ligand. Association with steroid hormone receptors depends on prior receptor activation--i.e., release from heat shock proteins. The sequence identified here appears to be a general partner protein for nuclear hormone receptors, with the gene being expressed in a variety of mammalian tissues.

Specific mutations in the ligand binding domain selectively abolish the silencing function of human thyroid hormone receptor beta.

Although most nuclear hormone receptors are ligand-dependent transcriptional activators, certain members of this superfamily, such as thyroid hormone receptor (TR) and retinoic acid receptor (RAR), are involved in transcriptional repression. The silencing function of these receptors has been localized to the ligand binding domain (LBD). Previously, we demonstrated that overexpression of either the entire LBD or only the N-terminal region of the LBD (amino acids 168-259) is able to inhibit the silencing activity of TR. From this result we postulated the existence of a limiting factor (corepressor) that is necessary for TR silencing activity. To support this hypothesis, we identified amino acids in the N-terminal region of the LBD of TR that are important for the corepressor interaction and for the silencing function of TR. The silencing activity of TR was unaffected by overexpression of the LBD of mutant TR (V174A/D177A), suggesting that valine at position 174 and/or aspartic acid at position 177 are important for corepressor interaction. This mutant receptor protein, V174/D177, also lost the ability to silence target genes, suggesting that these amino acids are important for silencing function. Control experiments indicate that this mutant TR maintains its wild-type hormone binding and transactivation functions. These findings further strengthen the idea that the N-terminal region of the LBD of TR interacts with a putative corepressor protein(s) to achieve silencing of basal gene transcription.