Christian Nanoff

Recruitment of a cytoplasmic chaperone relay by the A2A-adenosine receptor.

Background: The A2A receptor is known to accumulate in the endoplasmic reticulum. Results: Mass spectrometry identified molecular chaperones (HSP90 and HSP70) bound to the A2A receptor. Conclusion: Sequential recruitment of chaperones to the cytosolic face of the A2A receptor is consistent with a heat-shock protein relay assisting folding. Significance: The observations are consistent with a chaperone/COPII exchange model, where heat-shock proteins bound to the receptor preclude its premature ER export. The adenosine A2A receptor is a prototypical rhodopsin-like G protein-coupled receptor but has several unique structural features, in particular a long C terminus (of >120 residues) devoid of a palmitoylation site. It is known to interact with several accessory proteins other than those canonically involved in signaling. However, it is evident that many more proteins must interact with the A2A receptor, if the trafficking trajectory of the receptor is taken into account from its site of synthesis in the endoplasmic reticulum (ER) to its disposal by the lysosome. Affinity-tagged versions of the A2A receptor were expressed in HEK293 cells to identify interacting partners residing in the ER by a proteomics approach based on tandem affinity purification. The receptor-protein complexes were purified in quantities sufficient for analysis by mass spectrometry. We identified molecular chaperones (heat-shock proteins HSP90α and HSP70-1A) that interact with and retain partially folded A2A receptor prior to ER exit. Complex formation between the A2A receptor and HSP90α (but not HSP90β) and HSP70-1A was confirmed by co-affinity precipitation. HSP90 inhibitors also enhanced surface expression of the receptor in PC12 cells, which endogenously express the A2A receptor. Finally, proteins of the HSP relay machinery (e.g. HOP/HSC70-HSP90 organizing protein and P23/HSP90 co-chaperone) were recovered in complexes with the A2A receptor. These observations are consistent with the proposed chaperone/coat protein complex II exchange model. This posits that cytosolic HSP proteins are sequentially recruited to folding intermediates of the A2A receptor. Release of HSP90 is required prior to recruitment of coat protein complex II components. This prevents premature ER export of partially folded receptors.

A Two-state Model for the Diffusion of the A2A Adenosine Receptor in Hippocampal Neurons: AGONIST-INDUCED SWITCH TO SLOW MOBILITY IS MODIFIED BY SYNAPSE-ASSOCIATED PROTEIN 102 (SAP102)*

Background: Agonist activation slows diffusion of the A2A receptor in the lipid bilayer. Results: In hippocampal neurons, the agonist-induced decrease in mobility was accounted for by both the hydrophobic receptor core and its extended C terminus, which recruited SAP102. Conclusion: The observations are consistent with two diffusion states of the A2A receptor in neurons. Significance: SAP102 regulates access of the A2A receptor to a compartment with restricted mobility. The A2A receptor is a class A/rhodopsin-like G protein-coupled receptor. Coupling to its cognate protein, Gs, occurs via restricted collision coupling and is contingent on the presence of cholesterol. Agonist activation slows diffusion of the A2A adenosine receptor in the lipid bilayer. We explored the contribution of the hydrophobic core and of the extended C terminus by examining diffusion of quantum dot-labeled receptor variants in dissociated hippocampal neurons. Single particle tracking of the A2A receptor(1–311), which lacks the last 101 residues, revealed that agonist-induced confinement was abolished and that the agonist-induced decrease in diffusivity was reduced substantially. A fragment comprising the SH3 domain and the guanylate kinase domain of synapse-associated protein 102 (SAP102) was identified as a candidate interactor that bound to the A2A receptor C terminus. Complex formation between the A2A receptor and SAP102 was verified by coimmunoprecipitation and by tracking its impact on receptor diffusion. An analysis of all trajectories by a hidden Markov model was consistent with two diffusion states where agonist activation reduced the transition between the two states and, thus, promoted the accumulation of the A2A receptor in the compartment with slow mobility. Overexpression of SAP102 precluded the access of the A2A receptor to a compartment with restricted mobility. In contrast, a mutated A2A receptor (with 383DVELL387 replaced by RVRAA) was insensitive to the action of SAP102. These observations show that the hydrophobic core per se does not fully account for the agonist-promoted change in mobility of the A2A receptor. The extended carboxyl terminus allows for regulatory input by scaffolding molecules such as SAP102.

Comparison of the β-Adrenergic Receptor Antagonists Landiolol and Esmolol: Receptor Selectivity, Partial Agonism, and Pharmacochaperoning Actions.

Blockage of β1-adrenergic receptors is one of the most effective treatments in cardiovascular medicine. Esmolol was introduced some three decades ago as a short-acting β1-selective antagonist. Landiolol is a more recent addition. Here we compared the two compounds for their selectivity for β1-adrenergic receptors over β2-adrenergic receptors, partial agonistic activity, signaling bias, and pharmacochaperoning action by using human embryonic kidney (HEK)293 cell lines, which heterologously express each human receptor subtype. The affinity of landiolol for β1-adrenergic receptors and β2-adrenergic receptors was higher and lower than that of esmolol, respectively, resulting in an improved selectivity (216-fold versus 30-fold). The principal metabolite of landiolol (M1) was also β1-selective, but its affinity was very low. Both landiolol and esmolol caused a very modest rise in cAMP levels but a robust increase in the phosphorylation of extracellular signal regulated kinases 1 and 2, indicating that the two drugs exerted partial agonist activity with a signaling bias. If cells were incubated for ≥24 hours in the presence of ≥1 μM esmolol, the levels of β1-adrenergic—but not of β2-adrenergic—receptors increased. This effect was contingent on export of the β1-receptor from endoplasmic reticulum and was not seen in the presence of landiolol. On the basis of these observations, we conclude that landiolol offers the advantage of: 1) improved selectivity and 2) the absence of pharmacochaperoning activity, which sensitizes cells to rebound effects upon drug discontinuation.

Chaperoning of the A1-Adenosine Receptor by Endogenous Adenosine—An Extension of the Retaliatory Metabolite Concept

Cell-permeable orthosteric ligands can assist folding of G protein–coupled receptors in the endoplasmic reticulum (ER); this pharmacochaperoning translates into increased cell surface levels of receptors. Here we used a folding-defective mutant of human A1-adenosine receptor as a sensor to explore whether endogenously produced adenosine can exert a chaperoning effect. This A1-receptor-Y288A was retained in the ER of stably transfected human embryonic kidney 293 cells but rapidly reached the plasma membrane in cells incubated with an A1 antagonist. This was phenocopied by raising intracellular adenosine levels with a combination of inhibitors of adenosine kinase, adenosine deaminase, and the equilibrative nucleoside transporter: mature receptors with complex glycosylation accumulated at the cell surface and bound to an A1-selective antagonist with an affinity indistinguishable from the wild-type A1 receptor. The effect of the inhibitor combination was specific, because it did not result in enhanced surface levels of two folding-defective human V2-vasopressin receptor mutants, which were susceptible to pharmacochaperoning by their cognate antagonist. Raising cellular adenosine levels by subjecting cells to hypoxia (5% O2) reproduced chaperoning by the inhibitor combination and enhanced surface expression of A1-receptor-Y288A within 1 hour. These findings were recapitulated for the wild-type A1 receptor. Taken together, our observations document that endogenously formed adenosine can chaperone its cognate A1 receptor. This results in a positive feedback loop that has implications for the retaliatory metabolite concept of adenosine action: if chaperoning by intracellular adenosine results in elevated cell surface levels of A1 receptors, these cells will be more susceptible to extracellular adenosine and thus more likely to cope with metabolic distress.

The noradrenaline transporter as site of action for the anti-Parkinson drug amantadine

Amantadine is an established antiparkinsonian drug with a still unclear molecular site of action. Inxa0vivo studies on rodents, inxa0vitro studies on tissue of rodents as well as binding studies on post mortem human tissue implicate monoamine transporters and NMDA receptors. In order to re-examine its action at human variants of these proteins on intact cells we established cells stably expressing the human NR1/2A NMDA-receptor, noradrenaline transporter (NAT) or dopamine transporter (DAT) and tested the activity of amantadine in patch-clamp, uptake, release, and cytotoxicity experiments. Amantadine was less potent in blockade of NMDA-induced inward currents than in blockade of noradrenaline uptake and in induction of inward currents in NAT expressing cells. It was 30 times more potent in blocking uptake in NAT- than in DAT cells. Amantadine induced NAT-mediated release at concentrations of 10-100xa0μM in superfusion experiments and blocked NAT-mediated cytotoxicity of the parkinsonism inducing neurotoxin 1-methyl-4-phenyl-pyridinium (MPP(+)) at concentrations of 30-300xa0μM, whereas 300-1000xa0μM amantadine was necessary to block NMDA-receptor mediated cytotoxicity. Similar to amphetamine, amantadine was inactive at α(2A)-adrenergic receptors and induced reverse noradrenaline transport by NAT albeit with smaller effect size. Thus, amantadine acted as amphetamine-like releaser with selectivity for the noradrenergic system. These findings and differences with memantine, which had been reported as less efficient antiparkinsonian drug than amantadine but in our hands was significantly more potent at the NMDA-receptor, suggest contributions from a noradrenergic mechanism in the antiparkinsonian action of amantadine.

Sensitization of cAMP formation in a neuron-like cell line

Differentiation into a nerve cell-like phenotype and growth arrest of SH-SY5Y neuroblastoma cells went along with increased cAMP formation. Both receptor-dependent as well as direct activation of adenylyl cyclase by forskolin were enhanced by at least twenty-fold. Since cAMP controls many processes in nerve cell function and development we have investigated the causal factors and mechanism of sensitization in SH-SY5Y cells. The degree of sensitization depended on pre-incubation of the cells with retinoic acid; however, maximizing the extent of sensitization required the withdrawal of serum from the culture medium. This was necessary for the cells to secrete endogenous substances into the culture supernatant. Because sensitization was blocked by inhibitors of gene transcription we surmised that the autocrine factors were relevant for sensitization and were generated by de novo protein synthesis. A gene expression screen revealed several factor candidates (including dkk1, EphB2, NPY, VEGFB); our preliminary data indicated that a combination of these may be needed to induce full sensitization. Our data further suggest that sensitization was not due to up-regulation of stimulatory G proteins or adenylyl cyclase. Rather, the immediate cause may be clustering of the catalyst and its activator Gs. This interpretation is consistent with the effect caused by altering the membrane lipid composition which enhanced and reduced cAMP formation in undifferentiated and differentiated cells, respectively. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

The value of [11C]-acetate PET and [18F]-FDG PET in hepatocellular carcinoma before and after treatment with transarterial chemoembolization and bevacizumab

PurposeThis prospective study was to investigate the value of [11C]-acetate PET and [18F]-FDG PET in the evaluation of hepatocellular carcinoma (HCC) before and after treatment with transarterial chemoembolization (TACE) and vascular endothelial growth factor (VEGF) antibody (bevacizumab).MethodsTwenty-two patients (three women, 19 men; 62xa0±xa08xa0years) with HCC verified by histopathology were treated with TACE and bevacizumab (nxa0=xa011) or placebo (nxa0=xa011). [11C]-acetate PET and [18F]-FDG PET were performed before and after TACE with bevacizumab or placebo. Comparisons between groups were performed with t-tests and Chi-squared tests, where appropriate. Overall survival (OS) was defined as the time from start of bevacizumab or placebo until the date of death/last follow-up, respectively.ResultsThe patient-related sensitivity of [11C]-acetate PET, [18F]-FDG PET, and combined [11C]-acetate and [18F]-FDG PET was 68%, 45%, and 73%, respectively. There was a significantly higher rate of conversion from [11C]-acetate positive lesions to negative lesions in patients treated with TACE and bevacizumab as compared with that in patients with TACE and placebo (pxa0<xa00.05). In patients with negative acetate PET, the mean OS in patients treated with TACE and bevacizumab was 259xa0±xa0118xa0days and was markedly shorter as compared with that (668xa0±xa0217xa0days) in patients treated with TACE and placebo (pxa0<xa00.05). In patients treated with TACE and placebo, there was significant difference in mean OS in patients with positive FDG PET as compared with that in patients with negative FDG PET (pxa0<xa00.05). The HCC lesions had different tracer avidities showing the heterogeneity of HCC.ConclusionsOur study suggests that combining [18F]-FDG with [11C]-acetate PET could be useful for the management of HCC patients and might also provide relevant prognostic and molecular heterogeneity information.

Pharmacochaperoning of the ER-retained A1 adenosine receptor

Background The A1 adenosine receptor is a member of the rhodopsinrelated subfamily of GPCRs. Point mutations in the conserved NPxxY(x)5,6F motif at the junction of helix 7 and the C-terminus disrupt surface targeting of the receptor and result in its intracellular retention. This trafficking arrest can be overcome by addition of receptor ligands (pharmacochaperoning) that stabilize the receptor fold and thus promote surface expression. The mutants serve as a tool to explore a ramification of the retaliatory metabolite complex: hypoxia leads to intracellular accumulation of adenosine (by breakdown of ATP and by inhibition of adenosine kinase). Intracellular and extracellular adenosine levels are in equilibrium because of the action of the equilibrative nucleoside transporters. Extracellular adenosine dampens cellular metabolism by acting on inhibitory A1 adenosine receptors and thus counteracts the impact of hypoxia. If adenosine also pharmacochaperoned A1 adenosine receptors during hypoxia, it would enhance its effectiveness as a protective agent.

Autocrine signalling as cause of sensitized cAMP formation

Results SH-SY5Y cells (a model nerve cell) required differentiation to produce cAMP in substantial amounts; in undifferentiated proliferating cells, forskolin or activation of Gs-coupled receptors barely stimulated cAMP formation. A cell-autonomous process induced sensitization. The process relied on an autocrine factor, which we identified as Dickkopf1 protein. Serum protein quenched the activity of Dickkopf1; conversely, serum deprivation allowed for sensitization to unfold. The effect of Dickkopf1 was mediated by a high-affinity receptor activated at concentrations of ≤1 nM. In accordance with its cognate function as Wnt antagonist, sensitization was a consequence of suppressing the canonical Wnt signaling pathway; the inhibitors of glycogen-synthase kinase-3b, lithium chloride and, in addition, valproic acid mimicked Wnt signals and diminished the extent of sensitized cAMP formation. We found that in differentiated cells, expression of the a-subunit of Gs (Gas) increased due to activation of the GNAS gene. Although sufficient to support Gs-coupling of the A2A adenosine receptor, increased Gas alone failed to enhance receptor-stimulated cAMP formation. We infer that sensitized cAMP formation reflected increased responsiveness of the catalyst, adenylyl cyclase, to stimuli.

The folding interactome of GPCRs

Background The A2A adenosine receptor is a prototypical G proteincoupled receptor. It is expressed in a wide variety of cells including as different types as neurons, platelets, cells of the immune system and muscle. The A2A receptor has an unusually long C-terminus (of >120 residues), which for the most part is dispensable for coupling to Gs. This C-terminus turned out to be the docking site for other proteins. Using a yeast-2-hybrid screen we have previously identified proteins interacting with the C-terminus including ARNO/cytohesin2, SAP102 and USP4.