The A(2A)-adenosine receptor: a GPCR with unique features?

The A(2A)-adenosine receptor is a prototypical G(s)-coupled receptor. However, the A(2A)-receptor has several structural and functional characteristics that make it unique. In contrast to the classical model of collision coupling described for the beta-adrenergic receptors, the A(2A)-receptor couples to adenylyl cyclase by restricted collision coupling and forms a tight complex with G(s). The mechanistic basis for this is not clear; restricted collision coupling may arise from the interaction of the receptor with additional proteins or due to the fact that G protein-coupling is confined to specialized membrane microdomains. The A(2A)-receptor has a long C-terminus (of >120 residues), which is for the most part dispensable for coupling to G(s). It was originally viewed as the docking site for kinases and the beta-arrestin family to initiate receptor desensitization and endocytosis. The A(2A)-receptor is, however, fairly resistant to agonist-induced internalization. Recently, the C-terminus has also been appreciated as a binding site for several additional 'accessory' proteins. Established interaction partners include alpha-actinin, ARNO, USP4 and translin-associated protein-X. In addition, the A(2A)-receptor has also been reported to form a heteromeric complex with the D(2)-dopamine receptor and the metabotropic glutamate receptor-5. It is clear that (i) this list cannot be exhaustive and (ii) that all these proteins cannot bind simultaneously to the receptor. There must be rules of engagement, which allow the receptor to elicit different biological responses, which depend on the cellular context and the nature of the concomitant signal(s). Thus, the receptor may function as a coincidence detector and a signal integrator.

p21cip1 is required for the differentiation of oligodendrocytes independently of cell cycle withdrawal.

Differentiation of most cell types requires both establishment of G1 arrest and the induction of a program related to achieving quiescence. We have chosen to study the differentiation of oligodendrocyte cells to determine the role of p27 and p21 in this process. Here we report that both p27 and p21 are required for the appropriate differentiation of these cells. p27 is required for proper withdrawal from the cell cycle, p21 is not. Instead, p21 is required for the establishment of the differentiation program following growth arrest. Similar observations were made in vivo. We show that p21-/- cells withdraw from the cell cycle similar to wild-type cells; however, early in animal life, the brain is hypomyelinated, inferring that the loss of p21 delayed myelination in the cerebellum. We found that we could complement or bypass the differentiation failure in p21-/- cells with either PD98059, an inhibitor of Mek1, or by transducing them with a tat-p16ink4a protein. We concluded that the two cdk inhibitors serve non-redundant roles in this program of differentiation, with p27 being responsible for arrest and p21 having a function in differentiation independent of its ability to control exit from the cell cycle.

The cyclin-dependent kinase inhibitor p21cip1 mediates the growth inhibitory effect of phorbol esters in human venous endothelial cells.

Long-term application of the phorbol ester phorbol 12,13-dibutyrate (PDBu) inhibits the proliferation of human venous endothelial cells. The cyclin-dependent kinase inhibitor p21cip1 is a potential candidate mediating the PDBu-induced delayed entry of the cells into S-phase (by approximately 10 h when compared with cells stimulated with basic fibroblast growth factor (bFGF)). Levels of p21cip1 (protein and mRNA) rapidly rise (within approximately 2 h) in endothelial cells treated with the active isomer beta-PDBu, but not with alpha-PDBu; this effect is blocked by the mitogen-activated protein kinase kinase-1 (Mek1) inhibitor PD098059 and by the protein kinase C (PKC) antagonists GF109203X and rottlerin (selective for PKC-delta), but not Gö 6976 (selective for Ca2+-dependent PKC isoforms). Rapamycin blocks the PDBu-induced accumulation of p21cip1 (but not of the cognate mRNA), indicating an action of PKC on p21(cip1) mRNA translation. If endothelial cells are recruited into the cell cycle by bFGF, p21cip1 mRNA and protein levels rise initially (within 2 h) and decline subsequently such that p21cip1 drops to a minimum prior to the initiation of DNA synthesis (i.e. after approximately 12 h). In bFGF-stimulated cells, changes in p21cip1 mRNA and protein are strictly linked. In contrast, the levels of p21cip1 mRNA decline substantially (>10 h) before the protein decreases in PDBu-stimulated cells. Thus, PKC (presumably PKC-delta) regulates the amounts of p21cip1 in endothelial cells at the level of mRNA accumulation and translation, leading to a rapid and robust induction; following persistent PKC activation, p21(cip1) remains elevated despite reduced mRNA levels, indicating an enhanced stability of the protein. The bFGF-mediated increase in p21cip1 is blocked by the Mek1 inhibitor, but not by GF109203X; hence, in endothelial cells, induction of p21cip1 by PKC- and growth factor-dependent signaling is achieved by distinct pathways that converge and require activation of the mitogen-activated protein kinase cascade. The beta-PDBu-induced delayed S-phase entry and drop in p21cip1 are reversed if GF109203X is added 4 h after beta-PDBu to prevent persistent PKC activation. These observations indicate a cause and effect relation between sustained p21cip1 elevations and the delay in S-phase entry induced by beta-PDBu.

Interaction of allosteric ligands with GABAA receptors containing one, two, or three different subunits.

The presence of allosteric binding sites on recombinant GABAA receptors formed after transfection of human embryonic kidney (HEK) 293 cells with alpha 1-, beta 3-, or gamma 2-subunits, or with various combinations of these subunits, was systematically investigated. From all possible subunit combinations, high affinity [3H]muscimol binding sites were induced in cells transfected with alpha 1 beta 3- or alpha 1 beta 3 gamma 2-subunits only. GABAA receptor associated [3H]flunitrazepam binding sites were induced in cells after transfection with alpha 1 gamma 2- or alpha 1 beta 3, gamma 2-subunits, and [35S]r-butylbicyclophosphorothionate (TBPS) binding sites were found in cells transfected with beta 3-, beta 3 gamma 2-, alpha 1 beta 3-, or alpha 1 beta 3 gamma 2-subunits. Binding of [35S]TBPS could be inhibited by pentobarbital, etazolate, (+)-etomidate, alphaxalone, propofol, chlormethiazole, and 4'-chlorodiazepam (Ro 5-4864) with a potency which differed in cells transfected with beta 3-, beta 3 gamma 2-, alpha 1 beta 3-, or alpha 1 beta 3 gamma 2-subunits. Results obtained indicate that receptors with different subunit composition actually can be formed in HEK cells and exhibit distinct pharmacological properties.

Allosteric modulation of [3H]flunitrazepam binding to recombinant GABAA receptors.

The allosteric modulation of [3H]flunitrazepam binding by gamma-aminobutyric acid (GABA), pentobarbital, (+)-etomidate, etazolate, alphaxalone, propofol and chlormethiazole was investigated in cerebellar membranes and membranes from human embryonic kidney (HEK) 193 cells transfected with alpha 1 beta 3 gamma 2 or alpha 1 gamma 2 subunits. Results obtained indicate that [3H]flunitrazepam binding to recombinant GABAA receptors consisting of alpha 1 beta 3 gamma 2 subunits could be modulated by these compounds in a way and with a potency similar to that observed in cerebellar membranes. In addition, it was demonstrated that not only receptors consisting of alpha 1 beta 3 gamma 3, but also those consisting of alpha 1 gamma 2 subunits exhibited [3H]flunitrazepam binding which could be stimulated by GABA. In contrast to alpha 1 beta 3 gamma 2 receptors, however, [3H]flunitrazepam binding to recombinant alpha 1 gamma 2 receptors was inhibited by pentobarbital, (+)-etomidate, etazolate, alphaxalone, propofol and chlormethiazole. This seems to indicate that binding sites for these compounds are present on alpha 1 gamma 2 receptors, but that their allosteric interaction with [3H]flunitrazepam binding sites is different from that of alpha 1 beta 3 gamma 2 receptors.

Rat beta 3 subunits expressed in human embryonic kidney 293 cells form high affinity [35S]t-butylbicyclophosphorothionate binding sites modulated by several allosteric ligands of gamma-aminobutyric acid type A receptors.

Human embryonic kidney 293 cells transiently transfected with beta 3 subunits of gamma-aminobutyric acid type A receptors from the rat exhibited a specific high affinity binding for [35S]t-butylbicyclophosphorothionate (TBPS) that could be inhibited by pentobarbital, etazolate, (+)-etomidate, alphaxalone, propofol, chlormethiazole, and Ro 5-4864. The potency of these compounds for inhibition of [35S]TBPS binding was similar in membranes from beta 3 subunit-transfected human embryonic kidney 293 cells and in cerebellar membranes. In contrast to maximally inhibiting concentrations of unlabeled TBPS or picrotoxin, which caused a monophasic and rather slow dissociation of [35S]TBPS, maximally inhibiting concentrations of pentobarbital, etazolate, alphaxalone, propofol, chlormethiazole, and Ro 5-4864 accelerated the dissociation of [35S]TBPS from beta 3 subunit-containing membranes. Immunoaffinity chromatography and Western blot analysis with subunit-specific antibodies indicated that other endogenous subunits possibly present in these cells were not associated with beta 3 subunits. These results appear to indicate that most of the allosteric binding sites present on gamma-aminobutyric acid type A receptors can be formed by the beta subunit of these receptors. Homo-oligomeric beta 3 receptors therefore are an excellent model system for the structural investigation of gamma-aminobutyric acid type A receptors.

[3H]propyl-6-azido-beta-carboline-3-carboxylate: a new photoaffinity label for the GABAA-benzodiazepine receptor.

[3H]Propyl-6-azido-beta-carboline-3-carboxylate ([3H]ACCP) exhibited a high affinity for GABAA receptors affinity purified from the brains of adult rats, and binding of this compound could be inhibited by several ligands of the benzodiazepine binding site of GABAA receptors. On irradiation with UV light, [3H]ACCP, similarly to [3H]flunitrazepam, irreversibly labeled a protein with an apparent molecular weight of 51 kDa in affinity-purified GABAA receptors, and this labeling could be inhibited in the presence of diazepam. These data indicate that [3H]ACCP can be used as a photoaffinity label for GABAA receptors.

Endogenous [3H]flunitrazepam binding in human embryonic kidney cell line 293.

Specific endogenous [3H]flunitrazepam binding sites were identified and characterized in membranes from the human embryonic kidney (HEK) cell line 293. A large part of these binding sites exhibited an intermediate affinity for [3H]flunitrazepam and a microM affinity for diazepam, clonazepam, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide (PK 11195) or 4'-chlorodiazepam (Ro 5-4864). These sites, thus, resembled neither gamma-aminobutyric acidA (GABAA) receptor associated nor 'peripheral' benzodiazepine binding sites. A small part of the binding sites labeled by [3H]flunitrazepam seemed to belong to 'peripheral' benzodiazepine binding sites exhibiting a nM affinity for PK 11195, and another small part of the binding sites seemed to exhibit a high affinity for flunitrazepam and PK 11195. Although small amounts of mRNA for alpha 1-, beta 3- and gamma 2-subunits of GABAA receptors could be identified in HEK 293 cells, neither the actual expression of GABAA receptors in these cells nor a coassembly of endogenous subunits with transfected GABAA receptor subunits could be demonstrated by binding studies.

Cyclosporin A reduces skin collagen content in renal graft recipients.

Stimulated by the observation of a direct cytopathic effect of cyclosporin A on dermal fibroblasts we have examined total skin collagen content and collagenase activity in three groups of patients. Group 1 (controls) consisted of 16 patients without internal diseases, group 2 of 12 patients with renal transplantation on cyclosporin A therapy and group 3 of six patients with renal transplantation on corticosteroid/azathioprine therapy.Total skin collagen was measured by hydroxyproline/protein determination, collagenase activity according to the principle of Wünsch. SDS page was employed in order to show collagen split products.Mean skin collagen content (expressed by hydroxyproline/protein) was significantly lower in patients on cyclosporin A treatment (42.4 ± 12.2µg/mg) compared to controls (78.6 ± 14.2µg/mg) and patients on corticosteroid/azathioprine therapy (73.7 ± 11.2µg/mg). Mean collagenase activity was significantly higher in patients on cyclosporin A treatment (0.59 ± 0.16 IU) compared to controls (0.21 ± 0.09 IU) and patients on corticosteroid/azathioprine treatment (0.25 ± 0.11 IU). Total skin collagen content and collagenase activity were significantly inversely correlated in patients on cyclosporin A treatment (r = -0.82,p < 0.01,y = -0.011x + 1.053). Patients on cyclosporin A treatment showed remarkable reduction of alpha 1 and alpha 2 collagen chains and significantly prominent split products.The results of our study could be explained either by the activation of collagenase or as a consequence of cyclophilin (peptidyl-prolyl cis-trans-isomerase) inhibition.

Separation of alpha 1, alpha 2 and alpha 3 subunits of the GABAA-benzodiazepine receptor complex by immunoaffinity chromatography.

Polyclonal antibodies raised to synthetic amino acid sequences of the rat gamma-aminobutyric acid (GABAA) receptor alpha 1-, alpha 2- and alpha 3-subunits selectively recognized single proteins with apparent molecular weight 51 kDa (P51), 53 kDa (P53) and 59 kDa (P59), respectively, in GABAA receptor preparations affinity purified from the brains of 5-10-day-old rats. The antibodies were coupled to Affigel 10, and the resulting immunoaffinity columns were used to isolate these proteins from affinity purified GABAA receptors.

Isolation of type I and type II GABAA-benzodiazepine receptors by immunoaffinity chromatography.

Anti-peptide alpha 1 (1-9) and anti-peptide alpha 3 (459-467) antibodies coupled to Affigel-10 were used for the isolation of GABAA receptors containing the alpha 1- or alpha 3-subunit, respectively. Both types of GABAA receptors exhibited a high affinity for [3H]flunitrazepam, and binding of [3H]flunitrazepam was stimulated in the presence of GABA. GABAA receptors eluted from the anti-peptide alpha 1 (1-9) immunoaffinity column exhibited a high affinity and those from the anti-peptide alpha 3 (459-467) columns a low affinity for the type I benzodiazepine receptor-selective ligand Cl 218872, indicating the enrichment of type I and type II GABAA-benzodiazepine receptors, respectively.

Immunohistochemical localization of the alpha 1, alpha 2 and alpha 3 subunit of the GABAA receptor in the rat brain.

The immunohistochemical distribution of the alpha 1, alpha 2 and alpha 3 subunit of the gamma-aminobutyric acid-A (GABAA) receptor was investigated in the rat brain using affinity-purified antibodies against unique parts of the amino acid sequence of the respective subunits. The distribution of the 3 subunits differed markedly from each other indicating heterogeneity of the GABAA-receptor composition in different brain regions and at various receptive compartments (dendrites or somata) of neuronal cells.

Benzodiazepine receptor sites in the human brain: autoradiographic mapping.

Receptor autoradiography was used to localize and quantify the distribution of benzodiazepine receptor sites in human post mortem materials using [3H]flunitrazepam. The distribution and density of these sites was analysed in the brains of 21 patients dying without reported neurological disease. The distribution of benzodiazepine receptors in the human brain was found to be comparable from case to case although differences in the density occurred among the brains examined. No influence of the post mortem delay, age, gender or pre mortem drug treatment on the distribution and densities was observed in our series. The highest densities of benzodiazepine receptors in human brain were localized in cortical and hippocampal areas, nucleus accumbens, amygdala and mammillary bodies. Intermediate densities were found in the basal ganglia and thalamic and hypothalamic nuclei. [3H]Flunitrazepam binding was low in the brainstem nuclei and very low in white matter. The triazolopyridazine Cl 218872, reported to differentiate between type I and type II benzodiazepine receptor sites, exhibited regional differences in affinity when used to block [3H]flunitrazepam binding. Benzodiazepine receptors in the cerebellar cortex were more sensitive to this compound than those in the dentate gyrus of the hippocampus and the tuberal nuclei of the hypothalamus. An enrichment in the concentration of type I benzodiazepine receptor Cl 218872-sensitive sites was observed in motor areas as compared to structures of the limbic system. The addition of GABA to the incubation medium resulted in an increase of [3H]flunitrazepam binding, suggesting the coupling of these sites to a GABAA receptor. The increase in binding was directly proportional to the density of benzodiazepine receptors but unrelated to the density of high-affinity GABAA sites. The distribution of benzodiazepine receptor sites in the human brain compares well with that previously described in the rat brain. The high densities of receptors localized in the limbic system and in the cortical areas suggest that the effects of benzodiazepines are mediated through an interaction with the sites we have visualized in these anatomical structures. Our results provide a detailed map of the distribution of benzodiazepine receptors and a basis for the understanding of pharmacological effects of these drugs in humans and for future studies of modifications of these receptors in neurological and neuropsychiatric conditions in humans.

Comparison of tryptic peptides of benzodiazepine binding proteins photolabeled with [3H]flunitrazepam or [3H]Ro 15-4513.

When rat brain membranes were incubated with the benzodiazepine agonist [3H]flunitrazepam or the partial inverse benzodiazepine agonist [3H]Ro 15-4513 in the presence of ultraviolet light one protein (P51) was specifically and irreversibly labeled in cerebellum and at least two proteins (P51 and P55) were labeled in hippocampus. After digestion of the membranes with trypsin, protein P51 was degraded into several peptides. When P51 was photolabeled with [3H]Ro 15-4513, four peptides with apparent molecular weights of 39,000, 29,000, 21,000, and 17,000 were observed. When P51 was labeled with [3H]flunitrazepam, only two peptides with apparent molecular weights of 39,000 and 25,000 were obtained. Protein P55 was only partially degraded by trypsin, and whether it was labeled with [3H]flunitrazepam or [3H]Ro 15-4513 it yielded the same two proteolytic peptides with apparent molecular weights of 42,000 and 45,000. These results support the existence of at least two different benzodiazepine receptor subtypes associated with proteins P51 and P55. The different receptors seem to be differentially protected against treatment with trypsin. In addition, these results indicate that in the benzodiazepine receptor subtype associated with P51 benzodiazepine agonists and partial inverse benzodiazepine agonists irreversibly bind to different parts of the molecule.