The hydrophobic amino acid cluster at the cytoplasmic end of transmembrane helix III modulates the coupling of the ß(1)-adrenergic receptor to G(s).

Abstract A cluster of hydrophobic amino acids at the cytoplasmic end of trans-membranal helix III (TM-III) is a common feature among class-A of G protein-coupled receptors (GPCR). We mutagenized alanine 159(3.53) to glutamic acid and isoleucine160(3.54) to arginine (A159E/I160R) in TM-III of the human ß(1)-adrenergic receptor (ß(1)-AR) to disrupt the function of the hydrophobic cluster. Structurally, the combined mutations of A159E/I160R caused an almost 90° tilt in the rotation of Arg156(3.50) in the E/DRY motif of TM-III and displaced Tyr166(3.60) in intracellular loop 2. The A159E/I160R ß(1)-AR was uncoupled from G(s) as determined by cyclic AMP/adenylyl cyclase assays and by FRET-based proximity measurements between the ß(1)-AR and G(s)α. Isoproterenol induced ß-arrestin trafficking in cells expressing both the wild-type ß(1)-AR and the A159E/I160R ß(1)-AR. Isoproterenol markedly increased the phosphorylation of ERK1/2 in cells expressing the WT ß(1)-AR and this effect was dependent on the activation of the G(s)-cyclic AMP-dependent protein kinase → Rap → B-raf axis. However, in cells bearing the A159E/I160R ß(1)-AR, isoproterenol failed to increase the phosphorylation of ERK(1/2). These results indicate that mutations in the G(s)α-binding pocket of the GPCR interfered with receptor coupling to G(s) and with its downstream signaling cascades.

Rab11a and its binding partners regulate the recycling of the ß1-adrenergic receptor.

ß1-adrenergic receptors (ß1-AR) are internalized in response to agonists and then recycle back for another round of signaling. The serine 312 to alanine mutant of the ß1-AR (S312A) is internalized but does not recycle. We determined that WT ß1-AR and S312A were internalized initially to an early sorting compartment because they colocalized by >70% with the early endosomal markers rab5a and early endosomal antigen-1 (EEA1). Subsequently, the WT ß1-AR trafficked via rab4a-expressing sorting endosomes to recycling endosomes. In recycling endosomes WT ß1-AR were colocalized by >70% with the rab11 GTPase. S312A did not colocalize with either rab4a or rab11, instead they exited from early endosomes to late endosomes/lysosomes in which they were degraded. Rab11a played a prominent role in recycling of the WT ß1-AR because dominant negative rab11a inhibited, while constitutively active rab11a accelerated the recycling of the ß1-AR. Next, we determined the effect of each of the rab11-interacting proteins on trafficking of the WT ß1-AR. The recycling of the ß1-AR was markedly inhibited when myosin Vb, FIP2, FIP3 and rabphillin were knocked down. These data indicate that rab11a and a select group of its binding partners play a prominent role in recycling of the human ß1-AR.

Anxiolytic effects of phosphodiesterase-2 inhibitors associated with increased cGMP signaling.

Phosphodiesterase (PDE)-2 is a component of the nitric-oxide synthase (NOS)/guanylyl cyclase signaling pathway in the brain. Given recent evidence that pharmacologically induced changes in NO-cGMP signaling can affect anxiety-related behaviors, the effects of the PDE2 inhibitors (2-(3,4-dimethoxybenzyl)-7-det-5-methylimidazo-[5,1-f][1,2,4]triazin-4(3H)-one) (Bay 60-7550) and 3-(8-methoxy-1-methyl-2-oxo-7-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-5-yl)benzamide (ND7001), as well as modulators of NO, were assessed on cGMP signaling in neurons and on the behavior of mice in the elevated plus-maze, hole-board, and open-field tests, well established procedures for the evaluation of anxiolytics. Bay 60-7550 (1 microM) and ND7001 (10 microM) increased basal and N-methyl-d-aspartate- or detanonoate-stimulated cGMP in primary cultures of rat cerebral cortical neurons; Bay 60-7550, but not ND7001, also increased cAMP. Increased cGMP signaling, either by administration of the PDE2 inhibitors Bay 60-7550 (0.5, 1, and 3 mg/kg) or ND7001 (1 mg/kg), or the NO donor detanonoate (0.5 mg/kg), antagonized the anxiogenic effects of restraint stress on behavior in the three tests. These drugs also produced anxiolytic effects on behavior in nonstressed mice in the elevated plus-maze and hole-board tests; these effects were antagonized by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (20 mg/kg). By contrast, the NOS inhibitor N(omega)-nitro-l-arginine methyl ester (50 mg/kg), which reduces cGMP signaling, produced anxiogenic effects similar to restraint stress. Overall, the present behavioral and neurochemical data suggest that PDE2 may be a novel pharmacological target for the development of drugs for the treatment of anxiety disorders.

Chronic haloperidol and clozapine produce different patterns of effects on phosphodiesterase-1B, -4B, and -10A expression in rat striatum.

Phosphodiesterase-10A (PDE10A), -1B (PDE1B), -4B (PDE4B), and -4A (PDE4A) are important regulators of signal transduction in striatum due to their catalysis of cyclic AMP and cyclic GMP. The typical antipsychotic drug haloperidol and the atypical antipsychotic drug clozapine are thought to regulate cyclic nucleotide signaling in striatum. Since this brain region is essential in mediation of both therapeutic and extrapyramidal side effects, it was of interest to determine whether chronic treatment for 21 days with haloperidol (1 mg/kg) or clozapine (20 mg/kg) affected PDE expression in rat striatum. This was accomplished using SDS-PAGE/immunoblotting and real-time RT-PCR. Both antipsychotic drugs increased PDE10A and did not change PDE4A. By contrast, PDE1B was increased by haloperidol treatment, but not clozapine treatment, while PDE4B was increased by clozapine, but not haloperidol. In all cases, changes in protein expression were accompanied by corresponding changes in mRNA, and only were observed with chronic treatment. Up-regulation of PDEs may represent compensatory responses to haloperidol and clozapine treatments, due to altered cyclic nucleotide signaling, and that different patterns of effects produced by these drugs may be associated with their mechanisms of action.

Changes in NMDA receptor-induced cyclic nucleotide synthesis regulate the age-dependent increase in PDE4A expression in primary cortical cultures.

NMDA receptor-induced cAMP and cGMP are selectively hydrolyzed by PDE4 and PDE2, respectively, in rat primary cerebral cortical and hippocampal cultures. Because cAMP levels regulate the expression of PDE4 in rat primary cortical cultures, we examined the manner in which NMDA receptor activity regulates the age-dependent increase in the expression of PDE4A observed in vivo and in vitro. Inhibiting the activity of NR2B subunit with ifenprodil blocked NMDA receptor-induced cGMP synthesis and increased NMDA receptor-induced cAMP levels in a manner that reduced PDE4 activity. Therefore, NR1/NR2B receptor-induced cGMP signaling is involved in an acute cross-talk regulation of NR1/NR2A receptor-induced cAMP levels, mediated by PDE4. Chronic inhibition of NMDA receptor activity with MK-801 reduced PDE4A1 and PDE4A5 expression and activity in a time-dependent manner; this effect was reversed by adding the PKA activator dbr-cAMP. Inhibiting GABA receptors with bicuculline increased NMDA receptor-induced cAMP synthesis and PDE4A expression in cultures treated between DIV 16 and DIV 21 but not in cultures treated between DIV 8 and DIV 13. This effect was due to a high tone of NMDA receptor-induced cGMP in younger cultures, which negatively regulated the expression of PDE4A by a PKG-mediated process. The present results are consistent with behavioral data showing that both PDE4 and PDE2 are involved in NMDA receptor-mediated memory processes.

Regulation of phosphodiesterase-4 (PDE4) expression in mouse brain by repeated antidepressant treatment: comparison with rolipram.

Cyclic nucleotide phosphodiesterase-4 (PDE4) is a component of signaling pathways involved in the mediation of antidepressant activity. Of the four PDE4 subtypes, PDE4D appears to be of particular importance, given the finding that PDE4D-deficient mice exhibit an antidepressant-like behavioral phenotype. In mouse hippocampus and cerebral cortex, the effects of repeated treatment with the antidepressants desipramine and fluoxetine or the PDE4 inhibitor rolipram on the expression of PDE4D was compared to that of PDE4A and PDE4B, the other two subtypes expressed in the brain. Expression of PDE4D was increased by all drugs tested, with the exception of desipramine in hippocampus. By contrast, these treatments affected PDE4A and PDE4B expression differentially. In hippocampus, antidepressants increased PDE4A and decreased PDE4B, whereas ROL decreased PDE4A and did not change PDE4B. In cerebral cortex, antidepressants increased PDE4A and did not change PDE4B, whereas ROL did not change PDE4A and increased PDE4B. 3H-Rolipram binding was increased in cytosolic, but not in membrane, fractions of cerebral cortex by all drugs tested; there were no changes observed in hippocampus. Overall, the present results suggest some species-dependence of the regulation of PDE4 subtypes, based on data obtained previously using rats. They also suggest that the PDE4D subtype may be of particular importance as an antidepressant target in that it is regulated by repeated treatment with both norepinephrine and serotonin reuptake inhibitors as well as by the PDE4 inhibitor rolipram, drugs that produce antidepressant effects via different neuropharmacological mechanisms.