褪黑素受体与GABAA受体在褪黑素延长小鼠睡眠时间中的作用

目的：研究褪黑素受体和GABAA受体在褪黑素延长小鼠睡眠时间中的作用。方法：以翻正反射消失为睡眠开始的指标,至翻正反射恢复作为睡眠时间。观察不同受体激动剂或拮抗剂对褪黑素催眠作用的影响。结果：褪黑素3型受体拮抗剂盐酸哌唑嗪对褪黑素延长小鼠睡眠时间的作用无明显影响。GABA受体内源性激动剂GABA能明显增强褪黑素延长小鼠睡眠时间的作用,而GABAA受体上的印防己毒素结合位点的配基,即氯离子通道阻断剂印防己毒素能明显拮抗褪黑素的催眠作用,GABAA受体上的GABA结合位点的拮抗剂荷包牡丹碱则对褪黑素延长小鼠睡眠作用无明显影响。结论：褪黑素延长小鼠睡眠时间的作用与褪黑素3型受体无关,而与GABAA受体关系密切,其作用主要由印防己毒素结合位点介导。     

Modulation of steroid affinity for the androgen receptor by sucrose

The effects of sucrose on androgen binding to its receptor were investigated. Sucrose decreased the rate of thermal inactivation of unoccupied and occupied androgen receptor (AR) and the rates of [3H]5 alpha-dihydrotestosterone [( 3H]DHT) dissociation from both activated and nonactivated AR complexes. Binding of [3H]DHT to AR in vivo, or in intact cells at 37 degrees C, caused reduction of [3H]DHT dissociation from cytosolic and nuclear complexes, as compared to in vitro labeled receptor complexes. Further, exposure of these complexes to sucrose at 0 degrees C caused an additional reduction of dissociation rates. Thus, the decrease of [3H]DHT dissociation induced by sucrose is independent of the reaction that reduces DHT dissociation from activated and transformed AR. Sucrose also reduced the ability of mersalyl acid to inactivate AR complexes. This effect of sucrose was markedly diminished in the presence of 2M urea. Sucrose did not significantly affect the association rate, sedimentation properties, or nuclear binding ability of AR complexes, but it did decrease the equilibrium dissociation constant. Other monosaccharides and disaccharides also stabilized AR. These data suggest that sucrose induces conformational changes in the steroid binding domain of androgen receptor, thereby reducing the rates of inactivation, steroid dissociation, and the accessibility of sulfhydryl groups to mersalyl.     

Molecular defects of the androgen receptor

Defects of the androgen receptor cause a wide spectrum of abnormalities of phenotypic male development, ranging from individuals with mild defects of virilization to those with complete female phenotypes. In parallel with this phenotypic spectrum, a large number of different mutations have been identified that alter the synthesis or functional activity of the receptor protein. In many instances, the genetic mutations identified lead to an absence of the intact, full-length receptor protein. Such defects (splicing defects, termination codons, partial or complete gene deletions) invariably result in the phenotype of complete androgen insensitivity (complete testicular feminization). By contrast, single amino acid substitutions in the androgen receptor protein can result in the entire phenotypic spectrum of androgen resistant phenotypes and provide far more information on the functional organization of the receptor protein. Amino acid substitutions in different segments of the AR open-reading frame disturb AR function by distinct mechanisms. Substitutions in the DNA binding domain of the receptor appear to comprise a relatively homogeneous group. These substitutions impair the capacity of the receptor to bind to specific DNA sequence elements and to modulate the function of responsive genes. Amino acid substitutions in the hormone-binding domain of the receptor have a more varied effect on receptor function. In some instances, the resulting defect is obvious and causes an inability of the receptor to bind hormone. In other instances, the effect is subtler, and may result in the production of a receptor protein that displays qualitative abnormalities of hormone binding or from which hormone dissociates more rapidly. Often it is not possible to correlate the type of binding defect with the phenotype that is observed. Instead, it is necessary to measure the capacity of the receptor that is synthesized in functional assays in order to discern any type of correlation with phenotype. Finally, two types of androgen receptor mutation do not fit such a categorization. The first of these—the glutamine repeat expansion that is observed in spinal and bulbar muscular atrophy—leads to a reduction of receptor function that can be measured in heterologous cells or in fibroblasts established from such patients. The expression of ARs containing such expanded repeats in men is associated with a degeneration of motor neurons in the spinal cords of affected patients. Likewise, the alterations of androgen receptor structure that have been detected in advanced forms of prostate cancer also behave as gain-of-function mutations. In this latter type of mutation, the exquisite specificity of the normal androgen receptor is relaxed and the mutant receptors can be activated by a variety of steroidal and non-steroidal ligands.     

A review of the molecular conformations of melatonin ligands at the melatonin receptor

This review examines the 1992-2000 literature on studies of the molecular conformations of melatonin ligands at the melatonin receptor. In order to investigate quantitative structure-affinity relationships between different chemical classes of melatonergic ligands binding to the melatonin GPCR, CoMFA has been applied to extended sets of compounds, to obtain 3D-QSAR agonist/antagonist models. The results of several authors have suggested that the active conformation of the C-3 aminoethyl side chain of melatonin and related compounds is in a folded form, orthogonal to the aromatic ring. Positive steric potentials were found in the C-2 region, surrounding the C-5 methoxy group and near the N -acyl group of the side chain, while substituents in positions C-6 and C-7 cause a decrease in affinity. Negative steric regions were found between indole N-1 and C-2. Receptor binding affinities have been predicted for a range of structurally diverse compounds for the sheep brain melatonin receptor considering steric, electrostatic and lipophilic fields.     

Replacement of arginine 773 by cysteine or histidine in the human androgen receptor causes complete androgen insensitivity with different receptor phenotypes

We have discovered two different point mutations in a single codon of the X-linked androgen-receptor (AR) gene in two pairs of unrelated families who have complete androgen insensitivity (resistance) associated with different AR phenotypes in their genital skin fibroblasts. One mutation is a C-to-T transition at a CpG sequence near the 5' terminus of exon 6; it changes the sense of codon 773 from arginine to cysteine, ablates specific androgen-binding activity at 37 degrees C, and eliminates a unique KpnI site at the intron-exon boundary. The other mutation is a G-to-A transition that changes amino acid 773 to histidine and eliminates an SphI site. This mutant AR has a normal androgen-binding capacity at 37 degrees C but has a reduced affinity for androgens and is thermolabile in their presence. Transient transfection of COS cells with cDNA expression vectors yielded little androgen-binding activity at 37 degrees C from Arg773Cys and abundant activity with abnormal properties from Arg773His, thereby providing the pathogenicity of both sequence alterations. This conclusion coincides with the following facts about evolutionary preservation of the position homologous to Arg773 in the AR: it is occupied by Arg or lysine in the progesterone, glucocorticoid, and mineralocorticoid receptors, and it is within a 14-amino-acid region of their steroid-binding domains that share approximately 85% amino acid identity.     

Phosphorylation of the androgen receptor by a nuclear cAMP-independent protein kinase

The androgen receptor was purified from rat ventral prostate. The purified receptor migrated as a single band of mol. wt. 87000 on SDS-polyacrylamide gels, had a kd for R-1881 (17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one) binding as 6 nM, and sedimentation coefficient of 4.5 S. Phosphorylation of the purified receptor was studied by incubating it with [gamma-32P]ATP in the presence of several purified protein kinases including cAMP-dependent protein kinase, and four cAMP-independent protein kinases (which were active towards substrates such as phosvitin and casein). Phosphorylation of the 87000 mol. wt. androgen receptor protein occurred only in the presence of a nuclear cAMP-independent protein kinase (of the N2 type). No auto-phosphorylation of the receptor was detected. The results indicate that the androgen receptor is a phosphoprotein. Further, phosphorylation of the androgen receptor by only a specific nuclear cAMP-independent protein kinase may be important in determining the dynamics of its function.