Reddy Peera Kommaddi

Distinct roles for β-arrestin2 and arrestin-domain-containing proteins in β2 adrenergic receptor trafficking.

β‐arrestin 1 and 2 (also known as arrestin 2 and 3) are homologous adaptor proteins that regulate seven‐transmembrane receptor trafficking and signalling. Other proteins with predicted ‘arrestin‐like’ structural domains but lacking sequence homology have been indicated to function like β‐arrestin in receptor regulation. We demonstrate that β‐arrestin2 is the primary adaptor that rapidly binds agonist‐activated β2 adrenergic receptors (β2ARs) and promotes clathrin‐dependent internalization, E3 ligase Nedd4 recruitment and ubiquitin‐dependent lysosomal degradation of the receptor. The arrestin‐domain‐containing (ARRDC) proteins 2, 3 and 4 are secondary adaptors recruited to internalized β2AR–Nedd4 complexes on endosomes and do not affect the adaptor roles of β‐arrestin2. Rather, the role of ARRDC proteins is to traffic Nedd4–β2AR complexes to a subpopulation of early endosomes.

The p75NTR intracellular domain generated by neurotrophin-induced receptor cleavage potentiates Trk signaling.

The p75 neurotrophin receptor (p75NTR) potentiates Trk signaling, but the underlying mechanisms remain uncertain. Here, we examine the relationship between p75NTR cleavage and Trk signaling. We found that, in PC12 cells, nerve growth factor (NGF) induces rapid and robust α-secretase- and γ-secretase-dependent cleavage of p75NTR, releasing the resulting intracellular domain into the cytosol. Brain-derived neurotrophic factor similarly induces p75NTR cleavage in primary cerebellar granule neurons. p75NTR cleavage occurs by means of Trk-dependent activation of MEK-Erk signaling and induction of α-secretase activity, and is independent of ligand binding to p75NTR. Neurons and PC12 cells lacking p75NTR display defects in neurotrophin-dependent Akt activation. Normal Akt activation is rescued using full-length p75NTR or the p75 intracellular domain, but not cleavage-resistant p75NTR. We then demonstrate that NGF-dependent growth arrest of PC12 cells requires p75NTR cleavage and generation of the intracellular domain. We conclude that generation of the soluble p75NTR intracellular domain by Trk-induced cleavage plays a fundamental role in Trk-dependent signaling events.

Trk-dependent ADAM17 activation facilitates neurotrophin survival signaling

Signaling by TrkA and TrkB receptor tyrosine kinase is required for peripheral neuron survival. TrkA and TrkB signaling is facilitated by the p75 neurotrophin receptor (p75NTR), a member of the tumor necrosis factor (TNF) receptor superfam‐ily, through mechanisms that remain obscure. Here, we demonstrate that TrkA and TrkB induces MEK‐dependent phosphorylation of the transmembrane cysteine protease ADAM17 (a disintegrin and metal‐loprotease 17) at the intracellular residue threonine 735. Phosphorylation at this site activates ADAM17 and causes cleavage of p75NTR and production of the receptors’ intracellular domain (p75NTRICD) in PC12 cells and in primary cerebellar granule neurons. We show that Trk‐induced ADAM17 phosphorylation and generation of the p75NTR is required for neurotrophin‐induced Erk and Akt activation and for neurotrophin‐dependent survival signaling. Survival of PC12 cells maintained in 10 ng/ml nerve growth factor drops by 47% in cells depleted of ADAM17; this survival deficit is resolved if the p75NTR is overexpressed in the ADAM17 depleted cells. These studies identify a novel signaling circuit in which Trk activates ADAM17‐dependent p75NTRICD production to feedback to sustain Trk signaling and Trk‐dependent survival.—Kommaddi, R. P., Thomas, R., Ceni, C, Daigneault, K., Barker, P. A. Trk‐dependent ADAM17 activation facilitates neurotrophin survival signaling. FASEB J. 25, 2061‐2070 (2011). www.fasebj.org

Arrestins and protein ubiquitination.

The adaptor proteins, β-arrestins 1 and 2, were originally identified as inhibitors of G protein signaling at the seven-transmembrane receptors (7TMRs, also called G protein-coupled receptors or GPCRs). Subsequent studies have established β-arrestins as critical multifunctional 7TMR adaptors that mediate receptor trafficking and activate G protein-independent signaling pathways. 7TMR activation leads not only to the recruitment of arrestin proteins upon phosphorylation by GPCR kinases but also to β-arrestin ubiquitination. This posttranslational modification of β-arrestin is appended by specific E3 ubiquitin ligases and reversed by deubiquitinases, which are also recruited in a receptor- and agonist-specific manner. β-Arrestin ubiquitination allows it to form protein complexes with activated 7TMRs, endocytic proteins such as clathrin, and phosphorylated ERK1/2. β-Arrestin ubiquitination is dependent on its activated conformation and likely regulates timing and subcellular localization of various protein interactions during receptor trafficking and signaling. β-Arrestins also serve as adaptors that escort E3 ubiquitin ligases to mediate ubiquitination of a wide list of substrate proteins including 7TMRs and provide an added layer of regulation for defining substrate specificity in the cellular ubiquitination pathway.

Phosphorylation of the Deubiquitinase USP20 by Protein Kinase A Regulates Post-endocytic Trafficking of β2 Adrenergic Receptors to Autophagosomes during Physiological Stress

Background: The mechanisms for recruiting and activating deubiquitinase(s) during GPCR trafficking are unknown. Results: PKA phosphorylation of USP20 Ser-333 inhibits β2AR interaction as well as deubiquitination and promotes receptor degradation via autolysosomes during physiological stress. Conclusion: USP20 activity and substrate-specific interaction involves a phosphorylation code. Significance: We identify a novel role for PKA in USP20 regulation and ubiquitin-dependent sorting of GPCRs. Ubiquitination by the E3 ligase Nedd4 and deubiquitination by the deubiquitinases USP20 and USP33 have been shown to regulate the lysosomal trafficking and recycling of agonist-activated β2 adrenergic receptors (β2ARs). In this work, we demonstrate that, in cells subjected to physiological stress by nutrient starvation, agonist-activated ubiquitinated β2ARs traffic to autophagosomes to colocalize with the autophagy marker protein LC3-II. Furthermore, this trafficking is synchronized by dynamic posttranslational modifications of USP20 that, in turn, are induced in a β2AR-dependent manner. Upon β2AR activation, a specific isoform of the second messenger cAMP-dependent protein kinase A (PKAα) rapidly phosphorylates USP20 on serine 333 located in its unique insertion domain. This phosphorylation of USP20 correlates with a characteristic SDS-PAGE mobility shift of the protein, blocks its deubiquitinase activity, promotes its dissociation from the activated β2AR complex, and facilitates trafficking of the ubiquitinated β2AR to autophagosomes, which fuse with lysosomes to form autolysosomes where receptors are degraded. Dephosphorylation of USP20 has reciprocal effects and blocks trafficking of the β2AR to autophagosomes while promoting plasma membrane recycling of internalized β2ARs. Our findings reveal a dynamic regulation of USP20 by site-specific phosphorylation as well as the interdependence of signal transduction and trafficking pathways in balancing adrenergic stimulation and maintaining cellular homeostasis.

Stress‐induced expression of the p75 neurotrophin receptor is regulated by O‐GlcNAcylation of the Sp1 transcription factor

J. Neurochem. (2011) 116, 396–405.

Microgravity induces proteomics changes involved in endoplasmic reticulum stress and mitochondrial protection.

On Earth, biological systems have evolved in response to environmental stressors, interactions dictated by physical forces that include gravity. The absence of gravity is an extreme stressor and the impact of its absence on biological systems is ill-defined. Astronauts who have spent extended time under conditions of minimal gravity (microgravity) experience an array of biological alterations, including perturbations in cardiovascular function. We hypothesized that physiological perturbations in cardiac function in microgravity may be a consequence of alterations in molecular and organellar dynamics within the cellular milieu of cardiomyocytes. We used a combination of mass spectrometry-based approaches to compare the relative abundance and turnover rates of 848 and 196 proteins, respectively, in rat neonatal cardiomyocytes exposed to simulated microgravity or normal gravity. Gene functional enrichment analysis of these data suggested that the protein content and function of the mitochondria, ribosomes, and endoplasmic reticulum were differentially modulated in microgravity. We confirmed experimentally that in microgravity protein synthesis was decreased while apoptosis, cell viability, and protein degradation were largely unaffected. These data support our conclusion that in microgravity cardiomyocytes attempt to maintain mitochondrial homeostasis at the expense of protein synthesis. The overall response to this stress may culminate in cardiac muscle atrophy.

Aβ mediates F-actin disassembly in dendritic spines leading to cognitive deficits in Alzheimer's disease

Dendritic spine loss is recognized as an early feature of Alzheimers disease (AD), but the underlying mechanisms are poorly understood. Dendritic spine structure is defined by filamentous actin (F-actin) and we observed depolymerization of synaptosomal F-actin accompanied by increased globular-actin (G-actin) at as early as 1 month of age in a mouse model of AD (APPswe/PS1ΔE9, male mice). This led to recall deficit after contextual fear conditioning (cFC) at 2 months of age in APPswe/PS1ΔE9 male mice, which could be reversed by the actin-polymerizing agent jasplakinolide. Further, the F-actin-depolymerizing agent latrunculin induced recall deficit after cFC in WT mice, indicating the importance of maintaining F-/G-actin equilibrium for optimal behavioral response. Using direct stochastic optical reconstruction microscopy (dSTORM), we show that F-actin depolymerization in spines leads to a breakdown of the nano-organization of outwardly radiating F-actin rods in cortical neurons from APPswe/PS1ΔE9 mice. Our results demonstrate that synaptic dysfunction seen as F-actin disassembly occurs very early, before onset of pathological hallmarks in AD mice, and contributes to behavioral dysfunction, indicating that depolymerization of F-actin is causal and not consequent to decreased spine density. Further, we observed decreased synaptosomal F-actin levels in postmortem brain from mild cognitive impairment and AD patients compared with subjects with normal cognition. F-actin decrease correlated inversely with increasing AD pathology (Braak score, Aβ load, and tangle density) and directly with performance in episodic and working memory tasks, suggesting its role in human disease pathogenesis and progression. SIGNIFICANCE STATEMENT Synaptic dysfunction underlies cognitive deficits in Alzheimers disease (AD). The cytoskeletal protein actin plays a critical role in maintaining structure and function of synapses. Using cultured neurons and an AD mouse model, we show for the first time that filamentous actin (F-actin) is lost selectively from synapses early in the disease process, long before the onset of classical AD pathology. We also demonstrate that loss of synaptic F-actin contributes directly to memory deficits. Loss of synaptosomal F-actin in human postmortem tissue correlates directly with decreased performance in memory test and inversely with AD pathology. Our data highlight that synaptic cytoarchitectural changes occur early in AD and they may be targeted for the development of therapeutics.

Mdm2 regulates cardiac contractility by inhibiting GRK2-mediated desensitization of β-adrenergic receptor signaling

The oncoprotein Mdm2 is a RING domain-containing E3 ubiquitin ligase that ubiquitinates G protein-coupled receptor kinase 2 (GRK2) and β-arrestin2, thereby regulating β-adrenergic receptor (βAR) signaling and endocytosis. Previous studies showed that cardiac Mdm2 expression is critical for controlling p53-dependent apoptosis during early embryonic development, but the role of Mdm2 in the developed adult heart is unknown. We aimed to identify if Mdm2 affects βAR signaling and cardiac function in adult mice. Using Mdm2/p53-KO mice, which survive for 9-12 months, we identified a critical and potentially novel role for Mdm2 in the adult mouse heart through its regulation of cardiac β1AR signaling. While baseline cardiac function was mostly similar in both Mdm2/p53-KO and wild-type (WT) mice, isoproterenol-induced cardiac contractility in Mdm2/p53-KO was significantly blunted compared with WT mice. Isoproterenol increased cAMP in left ventricles of WT but not of Mdm2/p53-KO mice. Additionally, while basal and forskolin-induced calcium handling in isolated Mdm2/p53-KO and WT cardiomyocytes were equivalent, isoproterenol-induced calcium handling in Mdm2/p53-KO was impaired. Mdm2/p53-KO hearts expressed 2-fold more GRK2 than WT. GRK2 polyubiquitination via lysine-48 linkages was significantly reduced in Mdm2/p53-KO hearts. Tamoxifen-inducible cardiomyocyte-specific deletion of Mdm2 in adult mice also led to a significant increase in GRK2, and resulted in severely impaired cardiac function, high mortality, and no detectable βAR responsiveness. Gene delivery of either Mdm2 or GRK2-CT in vivo using adeno-associated virus 9 (AAV9) effectively rescued β1AR-induced cardiac contractility in Mdm2/p53-KO. These findings reveal a critical p53-independent physiological role of Mdm2 in adult hearts, namely, regulation of GRK2-mediated desensitization of βAR signaling.

Isoform-specific hyperactivation of calpain-2 occurs presymptomatically at the synapse in Alzheimer’s disease mice and correlates with memory deficits in human subjects

Calpain hyperactivation is implicated in late-stages of neurodegenerative diseases including Alzheimer’s disease (AD). However, calpains are also critical for synaptic function and plasticity, and hence memory formation and learning. Since synaptic deficits appear early in AD pathogenesis prior to appearance of overt disease symptoms, we examined if localized dysregulation of calpain-1 and/or 2 contributes to early synaptic dysfunction in AD. Increased activity of synaptosomal calpain-2, but not calpain-1 was observed in presymptomatic 1 month old APPswe/PS1ΔE9 mice (a mouse model of AD) which have no evident pathological or behavioural hallmarks of AD and persisted up to 10 months of age. However, total cellular levels of calpain-2 remained unaffected. Moreover, synaptosomal calpain-2 was hyperactivated in frontal neocortical tissue samples of post-mortem brains of AD-dementia subjects and correlated significantly with decline in tests for cognitive and memory functions, and increase in levels of β-amyloid deposits in brain. We conclude that isoform-specific hyperactivation of calpain-2, but not calpain-1 occurs at the synapse early in the pathogenesis of AD potentially contributing to the deregulation of synaptic signaling in AD. Our findings would be important in paving the way for potential therapeutic strategies for amelioration of cognitive deficits observed in ageing-related dementia disorders like AD.