Receptor最新文献

The crystal structure of the catalytic subunit of cAMP-dependent protein kinase serves as a template for the catalytic core of all eukaryotic protein kinases. The various crystal structures are reviewed with particular emphasis on the numerous conserved residues that converge at the active site. The structures also reveal the importance of posttranslational modifications, including myristylation and phosphorylation.

Presented is a brief review of the role of ecdysteroid and juvenile hormone (JH) receptors in the regulation of insect larval molting and metamorphosis of the epidermis and the nervous system, using examples from Manduca sexta and Drosophila melanogaster. Ecdysteroids cause a molt by combining with the ecdysone receptor (EcR) and the ultraspiracle protein (both members of the steroid hormone receptor superfamily) to activate directly a number of regulatory genes whose products both repress ongoing gene expression and stimulate genes associated with the production of the new stage in a cascading fashion. The presence of the JH-JH receptor (JHR) ensures that the molt is to another larval stage. At metamorphosis in the absence of JH, a new isoform of EcR and some new ecdysteroid-induced regulatory factors appear in response to low ecdysteroid. The subsequent high ecdysteroid then can activate a new cascade, causing the pupal molt. Also, the quantity of the new EcR isoform in metamorphosing larval neurons during adult development is correlated with cell fate. The JHR found in larval tissues is a 29-kDa nuclear protein that specifically binds JH with high affinity and is dependent on the presence of JH during ecdysteroid rises for its continued synthesis. This JHR represents a new class of intranuclear hormone receptors since it has no known DNA-binding domains and has a discrete subnuclear localization different from that of the EcR. Its specific action is unknown.

Understanding of the complex responses to thyroid and retinoid hormones has been greatly advanced through the cloning of specific nuclear receptors. The receptors belong to a large family of intracellular proteins that include the steroid hormone receptors and many "orphan" receptors for which no specific ligands are known. Recent studies on the mechanisms of actions of thyroid hormone receptors (TRs) and retinoic acid receptors (RARs) has revealed a complex system of receptor interactions. In contrast to the steroid hormone receptors that function predominantly as homodimers, TRs and RARs require heterodimer formation with the retinoid X receptors (RXR) for efficient DNA binding and transcriptional activation. RXRs in addition can form homodimers in the presence of specific ligands such as 9-cis retinoic acid. RXR homodimers recognize a subset of retinoic acid responsive elements (RARE). The COUP-TF orphan receptors also bind certain RAREs as homodimers and can inhibit thereby RAR/RXR heterodimers as well as RXR homodimer activities. Thus, a complex network of receptor interaction has been unraveled that promises a better understanding of thyroid and retinoid hormone action and that may allow the design of more effective hormonal therapeutics.

Association of nonhormone binding proteins with steroid receptor protein as well as modifications of the receptor by ATP, PPi, pyridoxal 5'-phosphate, molybdate, and the large number of less-well-characterized factors are important for the modulation and control of signal transduction in the steroid-receptor systems. Each of the steps in the steroid-receptor system, that is, the steroid binding, nuclear binding, DNA binding, and transcriptional regulations of specific genes (glucocorticoid response elements; GREs), may be controlled by the receptor interactions with other proteins and modulators. During the study on the regulatory mechanism of glucocorticoid-receptor system, we found a new endogenous factor in rat liver cytosol that increases the binding of activated glucocorticoid-receptor complex (GRC) to nuclei in the presence of ATP, and we named this factor ATP-stimulated translocation promoter (ASTP). Recently, we have purified ASTP protein to homogeneity and characterized it. ASTP has an M(r) of 93,000, and is composed of two apparently identical subunits with M(r) of 48,000. The sedimentation coefficient of ASTP is 6.5S, and its isoelectric point is 4.5. ASTP increases the binding of activated GRC to nuclei and chromatin, but not to DNA. ASTP activity is dependent on the physiological concentration of ATP, although ASTP does not bind to ATP-agarose. Interestingly, ASTP can bind to the arginine-rich histones H3 and H4, to which activated GRC also binds. In this article, we will summarize the biochemical properties of this interesting protein (ASTP) and will discuss our thinking about the mode of action.

In the search for more active agonists and antagonists of human growth hormone-releasing hormone (hGH-RH), various analogs are being synthesized. In order to follow the binding affinity of these analogs, we have developed a sensitive in vitro radioreceptor assay for GH-RH based on binding of labeled [His1,Nle27]hGH-RH(1-32)NH2 to rat anterior pituitary membrane homogenates by adapting and modifying earlier methods. Scatchard analysis of saturation binding data demonstrated the presence of a single class of specific binding sites for GH-RH in membranes of rat anterior pituitaries with a Bmax of 33.3 +/- 5.2 fmol/mg protein and an apparent Kd of 0.19 +/- 0.02 nM. In displacement analyses, we compared the binding affinity of [His1,Nle27]hGH-RH(1-32)NH2 with its iodinated derivative. No significant differences were detected in IC50 concentrations ranging from 0.97 to 3.4 nM between labeled and nonlabeled hGH-RH analogs. These findings demonstrate the validity of the radioreceptor assay. To evaluate the biological activities of hGH-RH derivatives, we applied a sensitive, dispersed rat pituitary cell superfusion system. This dynamic in vitro system eliminates the drawbacks of the static pituitary cell culture. No differences were observed in biological activities of the iodinated and noniodinated hGH-RH analogs. GH-releasing activity obtained from the superfusion assay correlated well with GH-RH receptor binding affinity for all nonlabeled and labeled hGH-RH analogs examined. These two methods are fast, simple, and relatively inexpensive, and provide quantitative data on receptor affinities, biological activities, and hence structure-affinity and structure-activity relationships. Joint use of these two in vitro systems appears to be suitable for screening newly synthesized GH-RH analogs.

Oxysterols are potent regulators of enzymes of the de novo cholesterol biosynthetic pathway and do not require the LDL (low density lipoprotein):LDL receptor system for their regulatory actions. The search for an alternate transduction system led to the identification of an oxysterol binding protein. This cytosolic protein has been extensively characterized, purified, and cloned. Although it fulfills the pharmacologic criteria for an oxysterol receptor by binding to oxysterols with affinities corresponding to their regulatory potencies, its function in maintaining cholesterol homeostasis has not been determined. We have overexpressed the human oxysterol receptor in Spodoptera frugiperda cells using the Baculovirus system. The overexpressed protein binds oxysterols, but not cholesterol. The affinity for 25-hydroxycholesterol determined by competitive binding assay was 7.3 +/- 4.4 nM (mean +/- SD), and the relative affinities of several other oxysterols approximately corresponded to their potencies in cell systems. The expressed protein migrated as a single immunoreactive band on denaturing polyacrylamide gels with a molecular mass of 94 kDa. The molecular mass calculated from sucrose gradient centrifugation and gel filtration was 273 kDa for the 9.8S form, 217 kDa for the 7.8S form, and 184 kDa for the 6.6S form. However, velocity gradient centrifugation and heparin-sepharose chromatography each indicated that there were at least two fractions containing specific oxysterol binding. We conclude that we have successfully overexpressed the human oxysterol receptor and that biochemical analysis of the overexpressed protein provides evidence of interactions with other proteins. Further analysis of the overexpressed protein should provide clues regarding its role in maintaining cholesterol homeostasis.

The synthetic steroid RU486, which displays antiprogestin and antiglucocorticoid properties in different systems, inhibits cell growth in dexamethasone-sensitive H56 cells containing glucocorticoid receptors, as well as in dexamethasone-resistant S-H56-125 cells displaying a very low level of dexamethasone binding. In order to better understand the mechanism of the antiproliferative effect, the binding of RU486 to these hepatoma cells was examined. Results revealed the presence of two different kinds of binding sites for RU486 in dexamethasone-sensitive H56 cells whereas only one type of site was detected in the dexamethasone-resistant cells. These peculiar sites were also recognized by cortivazol during competition experiments. Thus, it seems that S-H56-125 cells contain an altered glucocorticoid receptor that binds RU486 and cortivazol but virtually not dexamethasone. The ability of RU486 to inhibit the growth of dexamethasone-resistant cells suggests this steroid may be used to treat tumor cells that develop glucocorticoid resistance after long-term treatment.

Protein-tyrosine phosphatases (PTPases) that dephosphorylate the active (autophosphorylated) form of the insulin receptor and attenuate its tyrosine kinase activity play an essential regulatory role in signaling mediated by the insulin receptor. PTPases also modulate signaling through postreceptor pathways by catalyzing the dephosphorylation of cellular substrates of the insulin receptor kinase, such as IRS-1, or other tyrosine-phosphorylated proteins along the cellular cascade of insulin action. Recent studies have provided important data regarding PTPase(s) in insulin-responsive tissues that may regulate various components of the insulin action pathway. Further studies in this area will enhance our understanding of the mechanisms involved in insulin signaling and clarify the potential involvement of PTPases in the pathophysiology of insulin-resistant disease states.

Arg-Gly-Asp-Ser (RGDS) 1, Arg-Val-Asp-Ser (RVDS) 2, Arg-dVal-Asp-Ser (R[d]VDS) 3, and a cyclic RGD peptide, cyclo S,R [H-Pen-Arg-Gly-Asp-Pen-Gly-OH] 4, were tested for their ability to antagonize GPIIb-IIIa function. The activities were found to fall in the order 4 >> 1 >> 3 > 2. Simulated annealing and molecular dynamics studies were carried out to estimate the most populated conformations of each molecule. The acyclic molecules 1-3 were found to populate a much wider range of conformations than the cyclic molecule 4. The backbones of all four molecules were found to approximate beta-turn structures in the most populated conformations. In 4 the beta-turn intramolecular hydrogen bond between C = O of the i residue (Arg) and NH of the i + 3 residue (Ser) did not appear to be present. The distance between the beta-carbons of the critical Arg and Asp groups was found to be shorter in 4 (average 7.98 A) than in the less active acyclic molecules (averages of 8.65-9.33 A).