Phosphorylation of AMPA receptors

The ionotropic α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor is densely distributed in the mammalian brain and is primarily involved in mediating fast excitatory synaptic transmission. Recent studies in both heterologous expression systems and cultured neurons have shown that the AMPA receptor can be phosphorylated on their subunits (GluR1, GluR2, and GluR4). All phosphorylation sites reside at serine, threonine, or tyrosine on the intracellular C-terminal domain. Several key protein kinases, such as protein kinase A, protein kinase C, Ca²⁺/calmodulin-dependent protein kinase II, and tyrosine kinases (Trks; receptor or nonreceptor family Trks) are involved in the site-specific regulation of the AMPA receptor phosphorylation. Other glutamate receptors (N-methyl-d-aspartate receptors and metabotropic glutamate receptors) also regulate AMPA receptors through a protein phosphorylation mechanism. Emerging evidence shows that as a rapid and short-term mechanism, the dynamic protein phosphorylation directly modulates the electrophysiological, morphological (externalization and internalization trafficking and clustering), and biochemical (synthesis and subunit composition) properties of the AMPA receptor, as well as protein-protein interactions between the AMPA receptor subunits and various intracellular interacting proteins. These modulations underlie the major molecular mechanisms that ultimately affect many forms of synaptic plasticity.     

Phosphorylation of ryanodine receptors

Both cardiac and skeletal muscle ryanodine receptors (RyRs) are parts of large complexes that include a number of kinases and phosphatases. These RyRs have several potential phosphorylation sites in their cytoplasmic domains, but the functional consequences of phosphorylation and the identity of the enzymes responsible have been subjects of considerable controversy. Hyperphosphorylation of Ser-2809 in RyR2 (cardiac isoform) and Ser-2843 in RyR1 (skeletal isoform) has been suggested to cause the dissociation of the FK506-binding protein (FKBP) from RyRs, producing "leaky channels," but some laboratories find no relationship between phosphorylation and FKBP binding. Also debated is the identity of the kinases that phosphorylate these serines: cAMP-dependent protein kinase (PKA) versus calmodulin kinase II (CaMKII). Phosphorylation of other targets of these kinases could also alter calcium homeostasis. For example, PKA also phosphorylates phospholamban (PLB), altering the Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) activity. This review summarizes the major findings and controversies associated with phosphorylation of RyRs.     

Phosphorylation of the liver X receptors

The liver X receptors (LXRs) function as nutritional sensors for cholesterol and have important roles in lipid metabolism, glucose homeostasis, and inflammation. We provide the first evidence that LXRs are phosphorylated proteins. Mutational analysis and metabolic labeling indicate LXRalpha is phosphorylated on serine 198 in the hinge region. This is a consensus target for the MAPK family. A phosphorylation-deficient mutant, LXRalpha S198A, remains nuclear and responds to ligands like the wild-type protein. The biological significance of LXR phosphorylation remains to be elucidated but could provide a novel mechanism for the regulation of LXR signaling pathways and cellular metabolism.     

Intracellular traffic of steroid hormone receptors

The signal responsible for the nuclear localization of the progesterone receptor has been characterized. It is a complex signal. The study of the mechanism of this nuclear localization has revealed that the receptor continuously shuttles between the nucleus and the cytoplasm. The receptor diffuses into the cytoplasm and is constantly and actively transported back into the nucleus. The same phenomenon exists for estradiol and glucocorticoid receptors. The mechanism of entry of proteins into the nucleus is well documented, whereas the mechanism of their outward movement into the cytoplasm is not understood. We have grafted different nuclear localization signals (NLSs) onto β-galactosidase and have studied the traffic of this protein using heterokaryons and microinjection experiments. We have demonstrated that the same NLSs are involved in both the inward and the outward movement of proteins through the nuclear membrane. These results suggest that the nucleocytoplasmic shuttling may be a general phenomenon for nuclear proteins that could possibly undergo modifications in the cytoplasm and exert some biological activities there. These conclusions also imply that at least part of the cellular machinery involved in the nuclear import of proteins may function bidirectionally. Using these techniques, we have shown that two major antiprogestins, RU486 and ZK98299, act at the same distal level of hormone action.     

The steroid hormone levels and receptors of steroid hormone receptors in rat muscles during physical exertion

It is shown that the testosterone content in skeletal muscles of female albino rats is 2-fold decreased, while the estradiol content-1.5-fold increased and progesteron content showed no changes after systematic physical exercises. The pharmacokinetic investigations showed that androgen half-life in the organism decreased from 8 to 5 h under physical exercises. The amount of androgen receptors in cytosol of skeletal muscles increases from 1.34 +/- 0.08 to 1.71 +/- 0.10 fmol/mg per 1 mg of protein. Kd is 0.40 +/- 0.03 and 0.48 +/- 0.08 nM, respectively. Sensitivity of the organism to hormonal signal play an important role in the metabolism regulation in skeletal muscles along with the hormonal content change during the organism adaptation to physical exercises.     

Steroids,steroid receptors and disease

The 672nd Biochemical Society meeting was held at Sussex University, UK, from 19 to 21 December 2000. One session was dedicated to the study of steroids and their receptors, and related diseases.     

Phosphorylation and recycling kinetics of G protein-coupled receptors

The rate of ligand-induced phosphorylation of the V2 and V1a vasopressin receptors was characterized in HEK 293 cells. Both receptors were phosphorylated predominantly by GRKs, and the V1a receptor was also phosphorylated by protein kinase C regardless of the presence or absence of ligand. Phosphorylation of the V1aR catalyzed by GRKs reached maximal values at the shortest measured time: 15 seconds, and decayed rapidly with a t1/2 of 6 min in the continuous presence of AVP. In agreement with the hypothesis that dephosphorylation must precede receptor recycling to the cell surface, the V1aR returned rapidly to the cell surface after removal of the hormone from the medium. Phosphate incorporation into the V2R proceeded at a slower pace, and the internalized phosphorylated receptor failed to recycle to the cell surface and retained its phosphate for a long time in the presence or absence of ligand. A single mutation in the carboxy terminus of the V2R accelerated de-phosphorylation of the protein and conferred recycling properties to the V2R. These experiments provided molecular evidence for the hypothesis that internalization is required for de-phosphorylation and recycling of reactivated G protein coupled receptors to the cell surface.     

Sex steroid receptors in endometrial cancer

Endometrial cancer contains estrogen and progestin receptor proteins. The receptor content inversely correlates with histologic differentiation. The correlation between receptor content and histologic subtype and stage of the disease may depend upon the degree of differentiation of the tumor. The status of the peritoneal cytology and depth of myometrial invasion by tumor do not correlate with the receptor status. In general, patients with receptor-rich tumors have a better prognosis than those without receptors and respond favorably to progestin therapy. The level of the receptor protein in the tumor may also influence survival.     

Huntington's disease and steroid hormone receptors

BACKGROUND: Steroid hormone receptors constitute a special group of receptors having a wide range of efficiency and distribution in the body. Androgen and estrogen receptors, and their expression in the body, are linked with attributes such as reproduction control and sexual behaviour, but their relation with behavioural models, perception, memory and stress remain unclear to date. PURPOSE: In this project we aim to focus on monitoring the expressive influence of steroid hormone receptors on embryonic tissues and subsequently, expand our study to include the expression on adult tissues such as the CNS and to monitor the developmental aspects and relations pertaining to neurodegenerative disorders, such as Huntington's disease. MATERIAL AND METHODS: We shall rely on immuno-histochemistry, immuno-fluorescence and RT-PCR methods for detecting steroid hormone receptors and Huntingtin-associated protein 1 in the embryonic and adult tissue. CONCLUSION: Mapping the expression of steroid receptors during development represents an essential step in the quest for further studies and monitoring of the expression in adult tissues.     

Phosphorylation of transfected wild type and mutated progesterone receptors

An expression vector encoding wild type or mutated forms of the rabbit progesterone receptor was transfected into COS-7 cells and phosphorylation was studied by incubation with 32Pi followed by specific immunoprecipitation. The features of phosphorylation of the wild type receptor were identical to those previously observed in uterine cells: there was a basal level of phosphorylation which was increased approximately 7-fold by incubation with the hormone. The hyperphosphorylated receptor had decreased electrophoretic mobility ("upshift"). These experiments thus showed that the presence of the receptor specific kinase is not restricted to the target cells. Cleavage of the receptor by hydroxylamine and cyanogen bromide, and use of receptor mutants deleted in the N-terminal region, showed the absence of any detectable phosphorylation downstream from amino acid 520 (thus in the DNA and steroid binding domains). The majority of the phosphorylation sites were localized between amino acids 166 and 520. This localization was similar for basal and hormone-induced phosphorylation. DNA binding and hormone-induced hyperphosphorylation were not directly related, since deletion of the first zinc finger provided a hyperphosphorylated receptor. We showed that the constitutive receptor (totally deleted in the steroid binding region) exhibited only a low basal level of phosphorylation, and antagonist RU 486-receptor complexes were found to be hyperphosphorylated, leading us to conclude that the active form of the receptor was not the hyperphosphorylated one. Moreover receptor down regulation and hormone-induced receptor hyperphosphorylation were two independent phenomena. Basal phosphorylation was observed for both cytoplasmic and nuclear mutants, whereas nuclear localization was necessary but not sufficient for hyperphosphorylation. Finally, the second finger region and the hormone binding domain, which are necessary for receptor hyperphosphorylation, may be involved in the hormonally induced increased affinity of the receptor toward its kinase.     

Oligodeoxynucleotide base recognition by steroid hormone receptors

Oligodeoxynucleotides covalently linked to cellulose were used as probes of the DNA-binding domains of mouse steroid holoreceptors. With uterine cytosol estrogen receptor (E₂R) the relative binding order, in prior studies, was oligo(dG) > oligo(dT) ≧ oligo(dC) > > oligo(dA) > oligo(dI). The binding reactions were salt-sensitive with an optimal KCl concentration of 0.1–0.2 M. There was no enhancement of binding by activation, either temperature- or salt-induced. In the present study, using the oligomer ligands at a lower concentration, oligo(dT) binding was greater than that to oligo(dC). Quantitative differences in oligodeoxynucleotide binding were elicited by a number of inhibitors. These differences are again seen by exposure of E₂R to chaotropic salts such as SCN^?, ClO₄^? and NO₃^? as well as to putative modifiers of receptor amino acids, ie, iodoacetamide, 1,2 cyclohexanedione, and Rose Bengal. These results, and the quantitative differences following heat and purification, led to a designation of two types of subsites within the DNA-binding domain of uterine E₂R. These are stable G sites, which interact with oligo(dG); and labile N sites, which bind to oligo(dT), oligo(dC) and oligo(dA). Stimulation of binding to N sites and stabilization of the holoreceptor was effected by histones H2A and H2B. However, the differential response to incubation at 37°C was not altered by addition of H2B. Treatment of uterine E₂R by limited proteolysis also eliminated the stimulatory response to H2B. The above data, as well as prior studies, indicate that steroid holoreceptors can discriminate between the structural features of deoxynucleotide bases and this recognition process can be modulated by accessory proteins.     

Sex steroid receptors in human lung diseases

Several epidemiological studies have reported that gender differences exist in clinical and biological manifestations of human lung diseases. In particular, women are far more likely to develop both neoplastic and non-neoplastic lung diseases than men. This gender difference above suggests that sex steroid may be involved in the pathogenesis of various lung diseases. These sex steroids mediate their effects through sex steroid receptors including estrogen receptors (ER) i.e. ERα and ERβ progesterone receptors (PR) i.e. PR-A and PR-B and androgen receptors (ARs), all of which have been reported to be expressed in lung tissue. Therefore it becomes important to clarify the potential roles of sex steroid receptor in both neoplastic and non-neoplastic lung diseases toward improved treatment options for the patients. In this review, we summarized a number of studies in humans and experimental animals that have identified possible roles of sex steroids in respiratory physiology and pathology.