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Sudha K Shenoy

Publications and source records attributed to Sudha K Shenoy.

17 recordsLinked to original sources
PubMed2006

Trafficking of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) play an integral role in the signal transduction of an enormous array of biological phenomena, thereby serving to modulate at a molecular level almost all components of human biology. This role is nowhere more evident than in cardiovascular biology, where GPCRs regulate such core measures of cardiovascular function as heart rate, contractility, and vascular tone. GPCR/ligand interaction initiates signal transduction cascades, and requires the presence of the receptor at the plasma membrane. Plasma membrane localization is in turn a function of the delivery of a receptor to and removal from the cell surface, a concept defined most broadly as receptor trafficking. This review illuminates our current view of GPCR trafficking, particularly within the cardiovascular system, as well as highlights the recent and provocative finding that components of the GPCR trafficking machinery can facilitate GPCR signaling independent of G protein activation.

Cardiovascular System↗
PubMed2005

Angiotensin II-stimulated signaling through G proteins and beta-arrestin.

Beta-arrestin, originally identified as a protein that inhibits heterotrimeric guanine nucleotide-binding protein (G protein) coupling to cognate seven-transmembrane receptors [(7TMRs), also known as G protein-coupled receptors (GPCRs)], is currently being appreciated as a positive signaling mediator for various cell surface receptors. Activation of mitogen-activated protein kinases (MAPKs), especially extracellular signal regulated kinases 1 and 2 (ERK1/2), is a hallmark of intracellular signaling resulting from stimulation of various growth factor receptors, as well as 7TMRs. The resulting ERK activity can occur through multiple parallel or converging mechanisms. Using human embryonic kidney 293 (HEK-293) cells as a model system and utilizing RNA interference technology, two distinct pathways of angiotensin II-mediated ERK activation have been uncovered: (i) a G protein-dependent pathway that produces a transient activation of nuclear ERK and (ii) a beta-arrestin-dependent pathway that leads to sustained activation of ERK that is localized to the cytosol and endosomes. The spatial and temporal segregation of ERK activated by G protein and beta-arrestin pathways suggests that the physiological consequences may be different, and thus ligands that selectively stimulate or inhibit one of these pathways may be therapeutically valuable.

Angiotensin II↗
PubMed2005

beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor.

Physiological effects of beta adrenergic receptor (beta2AR) stimulation have been classically shown to result from G(s)-dependent adenylyl cyclase activation. Here we demonstrate a novel signaling mechanism wherein beta-arrestins mediate beta2AR signaling to extracellular-signal regulated kinases 1/2 (ERK 1/2) independent of G protein activation. Activation of ERK1/2 by the beta2AR expressed in HEK-293 cells was resolved into two components dependent, respectively, on G(s)-G(i)/protein kinase A (PKA) or beta-arrestins. G protein-dependent activity was rapid, peaking within 2-5 min, was quite transient, was blocked by pertussis toxin (G(i) inhibitor) and H-89 (PKA inhibitor), and was insensitive to depletion of endogenous beta-arrestins by siRNA. beta-Arrestin-dependent activation was slower in onset (peak 5-10 min), less robust, but more sustained and showed little decrement over 30 min. It was insensitive to pertussis toxin and H-89 and sensitive to depletion of either beta-arrestin1 or -2 by small interfering RNA. In G(s) knock-out mouse embryonic fibroblasts, wild-type beta2AR recruited beta-arrestin2-green fluorescent protein and activated pertussis toxin-insensitive ERK1/2. Furthermore, a novel beta2AR mutant (beta2AR(T68F,Y132G,Y219A) or beta2AR(TYY)), rationally designed based on Evolutionary Trace analysis, was incapable of G protein activation but could recruit beta-arrestins, undergo beta-arrestin-dependent internalization, and activate beta-arrestin-dependent ERK. Interestingly, overexpression of GRK5 or -6 increased mutant receptor phosphorylation and beta-arrestin recruitment, led to the formation of stable receptor-beta-arrestin complexes on endosomes, and increased agonist-stimulated phospho-ERK1/2. In contrast, GRK2, membrane translocation of which requires Gbetagamma release upon G protein activation, was ineffective unless it was constitutively targeted to the plasma membrane by a prenylation signal (CAAX). These findings demonstrate that the beta2AR can signal to ERK via a GRK5/6-beta-arrestin-dependent pathway, which is independent of G protein coupling.

Amino Acid Sequence↗
PubMed2005

Seven-transmembrane receptor signaling through beta-arrestin.

Cell surface receptors are important communicators of external stimuli to the cell interior where they lead to initiation of various signaling pathways and cellular responses. The largest receptor family is the seven-transmembrane receptor (7TMR) family, with approximately 1000 coding genes in the human genome. When 7TMRs are stimulated with agonists, they activate heterotrimeric guanine nucleotide-binding proteins (G proteins), leading to the production of signaling second messengers, such as adenosine 3',5'-monophosphate, inositol phosphates, and others. Activated receptors are rapidly phosphorylated on serine and threonine residues by specialized enzymes called G protein-coupled receptor kinases. Phosphorylated receptors bind the multifunctional adaptor proteins beta-arrestin1 and beta-arrestin2 with high affinity. Beta-arrestin binding blocks further G protein coupling, leading to "desensitization" of G protein-dependent signaling pathways. For several years, this was considered the sole function of beta-arrestins. However, novel functions of beta-arrestins have been discovered. Beta-arrestins are now designated as important adaptors that link receptors to the clathrin-dependent pathway of internalization. Beta-arrestins bind and direct the activity of several nonreceptor tyrosine kinases in response to 7TMR stimulation. Beta-arrestins also bind and scaffold members of such signaling cascades as the mitogen-activated protein kinases (MAPKs). Beta-arrestins are crucial components in 7TMR signaling leading to cellular responses that include cell survival and chemotaxis. Beta-arrestins act as endocytic adaptors and signal mediators not only for the 7TMRs, but also for several receptor tyrosine kinases.

Animals↗
PubMed2005

{beta}-Arrestin is crucial for ubiquitination and down-regulation of the insulin-like growth factor-1 receptor by acting as adaptor for the MDM2 E3 ligase.

The insulin-like growth factor-1 receptor (IGF-1R) plays important roles in physiological growth and aging as well as promoting several crucial functions in cancer cells. However, the molecular mechanisms involved in expression and down-regulation of IGF-1R are still poorly understood. Here we provide evidence that beta-arrestin, otherwise known to be involved in the regulation of G protein-coupled receptors, serves as an adaptor to bring the oncoprotein E3 ubiquitin ligase MDM2 to the IGF-1R. In this way, beta-arrestin acts as a crucial component in the ubiquitination and down-regulation of the receptor. Both MDM2 and beta-arrestin co-immunoprecipitated with the IGF-1R. The beta-arrestin isoform 1 appeared to be more strongly associated with the receptor than isoform 2, and in a molecular context it was 4-fold more efficient in inducing polyubiquitination of IGF-1R, a reaction that required the presence of beta-arrestin and MDM2. Ligand stimulation accelerated IGF-1R ubiquitination. In mouse P6 cells (overexpressing human IGF-1R) absence of beta-arrestin 1, but not of beta-arrestin 2, blocked ubiquitination of IGF-1R. Conversely, in the two studied human melanoma cell lines both beta-arrestin isoforms seemed to be involved in IGF-1R ubiquitination. However, because depletion of beta-arrestin 1 almost completely eliminated degradation, and IGF-1 induced down-regulation of the receptor in these cells, whereas beta-arrestin 2 only had a partial effect, beta-arrestin 1 seems to the more important isoform in affecting the expression of IGF-1R. To our knowledge this is the first study demonstrating a defined molecular role of beta-arrestin with direct relevance to cell growth and cancer.

Animals↗
PubMed2005

Transduction of receptor signals by beta-arrestins.

The transmission of extracellular signals to the interior of the cell is a function of plasma membrane receptors, of which the seven transmembrane receptor family is by far the largest and most versatile. Classically, these receptors stimulate heterotrimeric G proteins, which control rates of generation of diffusible second messengers and entry of ions at the plasma membrane. Recent evidence, however, indicates another previously unappreciated strategy used by the receptors to regulate intracellular signaling pathways. They direct the recruitment, activation, and scaffolding of cytoplasmic signaling complexes via two multifunctional adaptor and transducer molecules, beta-arrestins 1 and 2. This mechanism regulates aspects of cell motility, chemotaxis, apoptosis, and likely other cellular functions through a rapidly expanding list of signaling pathways.

Animals↗
PubMed2005

Receptor-specific ubiquitination of beta-arrestin directs assembly and targeting of seven-transmembrane receptor signalosomes.

Angiotensin II type 1a (AT1a), vasopressin V2, and neurokinin 1 (NK1) receptors are seven-transmembrane receptors (7TMRs) that bind and co-internalize with the multifunctional adaptor protein, beta-arrestin. These receptors also lead to robust and persistent activation of extracellular-signal regulated kinase 1/2 (ERK1/2) localized on endosomes. Recently, the co-trafficking of receptor-beta-arrestin complexes to endosomes was demonstrated to require stable beta-arrestin ubiquitination (Shenoy, S. K., and Lefkowitz, R. J. (2003) J. Biol. Chem. 278, 14498-14506). We now report that lysines at positions 11 and 12 in beta-arrestin2 are specific and required sites for its AngII-mediated sustained ubiquitination. Thus, upon AngII stimulation the mutant beta-arrestin2(K11,12R) is only transiently ubiquitinated, does not form stable endocytic complexes with the AT1aR, and is impaired in scaffolding-activated ERK1/2. Fusion of a ubiquitin moiety in-frame to beta-arrestin2(K11,12R) restores AngII-mediated trafficking and signaling. Wild type beta-arrestin2 and beta-arrestin2(K11R,K12R)-Ub, but not beta-arrestin2(K11R,K12R), prevent nuclear translocation of pERK. These findings imply that sustained beta-arrestin ubiquitination not only directs co-trafficking of receptor-beta-arrestin complexes but also orchestrates the targeting of "7TMR signalosomes" to microcompartments within the cell. Surprisingly, binding of beta-arrestin2(K11R,K12R) to V2R and NK1R is indistinguishable from that of wild type beta-arrestin2. Moreover, ubiquitination patterns and ERK scaffolding of beta-arrestin2(K11,12R) are unimpaired with respect to V2R stimulation. In contrast, a quintuple lysine mutant (beta-arrestin2(K18R,K107R,K108R,K207R,K296R)) is impaired in endosomal trafficking in response to V2R but not AT1aR stimulation. Our findings delineate a novel regulatory mechanism for 7TMR signaling, dictated by the ubiquitination of beta-arrestin on specific lysines that become accessible for modification due to the specific receptor-bound conformational states of beta-arrestin2.

Active Transport, Cell Nucleus↗
PubMed2004

Activation-dependent conformational changes in {beta}-arrestin 2.

Beta-arrestins are multifunctional adaptor proteins, which mediate desensitization, endocytosis, and alternate signaling pathways of seven membrane-spanning receptors (7MSRs). Crystal structures of the basal inactive state of visual arrestin (arrestin 1) and beta-arrestin 1 (arrestin 2) have been resolved. However, little is known about the conformational changes that occur in beta-arrestins upon binding to the activated phosphorylated receptor. Here we characterize the conformational changes in beta-arrestin 2 (arrestin 3) by comparing the limited tryptic proteolysis patterns and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) profiles of beta-arrestin 2 in the presence of a phosphopeptide (V(2)R-pp) derived from the C terminus of the vasopressin type II receptor (V(2)R) or the corresponding nonphosphopeptide (V(2)R-np). V(2)R-pp binds to beta-arrestin 2 specifically, whereas V(2)R-np does not. Activation of beta-arrestin 2 upon V(2)R-pp binding involves the release of its C terminus, as indicated by exposure of a previously inaccessible cleavage site, one of the polar core residues Arg(394), and rearrangement of its N terminus, as indicated by the shielding of a previously accessible cleavage site, residue Arg(8). Interestingly, binding of the polyanion heparin also leads to release of the C terminus of beta-arrestin 2; however, heparin and V(2)R-pp have different binding site(s) and/or induce different conformational changes in beta-arrestin 2. Release of the C terminus from the rest of beta-arrestin 2 has functional consequences in that it increases the accessibility of a clathrin binding site (previously demonstrated to lie between residues 371 and 379) thereby enhancing clathrin binding to beta-arrestin 2 by 10-fold. Thus, the V(2)R-pp can activate beta-arrestin 2 in vitro, most likely mimicking the effects of an activated phosphorylated 7MSR. These results provide the first direct evidence of conformational changes associated with the transition of beta-arrestin 2 from its basal inactive conformation to its biologically active conformation and establish a system in which receptor-beta-arrestin interactions can be modeled in vitro.

Amino Acid Sequence↗
PubMed2004

Constitutive protease-activated receptor-2-mediated migration of MDA MB-231 breast cancer cells requires both beta-arrestin-1 and -2.

Protease-activated receptor-2 (PAR-2) is activated by trypsin-like serine proteases and can promote cell migration through an ERK1/2-dependent pathway, involving formation of a scaffolding complex at the leading edge of the cell. Previous studies also showed that expression of a dominant negative fragment of beta-arrestin-1 reduces PAR-2-stimulated internalization, ERK1/2 activation, and cell migration; however, this reagent may block association of many proteins, including beta-arrestin-2 with clathrin-coated pits. Here we investigate the role of PAR-2 in the constitutive migration of a metastatic breast cancer cell line, MDA MB-231, and use small interfering RNA to determine the contribution of each beta-arrestin to this process. We demonstrate that a trypsin-like protease secreted from MDA MB-231 cells can promote cell migration through autocrine activation of PAR-2 and this correlates with constitutive localization of PAR-2, beta-arrestin-2, and activated ERK1/2 to pseudopodia. Addition of MEK-1 inhibitors, trypsin inhibitors, a scrambled PAR-2 peptide, and silencing of beta-arrestins with small interfering RNA also reduce base-line migration of MDA MB-231 cells. In contrast, a less metastatic PAR-2 expressing breast cancer cell line does not exhibit constitutive migration, pseudopodia formation, or trypsin secretion; in these cells PAR-2 is more uniformly distributed around the cell periphery. These data demonstrate a requirement for both beta-arrestins in PAR-2-mediated motility and suggest that autocrine activation of PAR-2 by secreted proteases may contribute to the migration of metastatic tumor cells through beta-arrestin-dependent ERK1/2 activation.

Arrestins↗
PubMed2004

Differential kinetic and spatial patterns of beta-arrestin and G protein-mediated ERK activation by the angiotensin II receptor.

The seven-membrane-spanning angiotensin II type 1A receptor activates the mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) by distinct pathways dependent on either G protein (likely G(q)/G(11)) or beta-arrestin2. Here we sought to distinguish the kinetic and spatial patterns that characterize ERK1/2 activated by these two mechanisms. We utilized beta-arrestin RNA interference, the protein kinase C inhibitor Ro-31-8425, a mutant angiotensin II receptor (DRY/AAY), and a mutant angiotensin II peptide (SII-angiotensin), which are incapable of activating G proteins, to isolate the two pathways in HEK-293 cells. G protein-dependent activation was rapid (peak <2 min), quite transient (t((1/2)) approximately 2 min), and led to nuclear translocation of the activated ERK1/2 as assessed by confocal microscopy. In contrast, beta-arrestin2-dependent activation was slower (peak 5-10 min), quite persistent with little decrement noted out to 90 min, and entirely confined to the cytoplasm. Moreover, ERK1/2 activated via beta-arrestin2 accumulated in a pool of cytoplasmic endosomal vesicles that also contained the internalized receptors and beta-arrestin. Such differential regulation of the temporal and spatial patterns of ERK1/2 activation via these two pathways strongly implies the existence of distinct physiological endpoints.

Angiotensin II↗
PubMed2004

beta-arrestin-1 competitively inhibits insulin-induced ubiquitination and degradation of insulin receptor substrate 1.

beta-arrestin-1 is an adaptor protein that mediates agonist-dependent internalization and desensitization of G-protein-coupled receptors (GPCRs) and also participates in the process of heterologous desensitization between receptor tyrosine kinases and GPCR signaling. In the present study, we determined whether beta-arrestin-1 is involved in insulin-induced insulin receptor substrate 1 (IRS-1) degradation. Overexpression of wild-type (WT) beta-arrestin-1 attenuated insulin-induced degradation of IRS-1, leading to increased insulin signaling downstream of IRS-1. When endogenous beta-arrestin-1 was knocked down by transfection of beta-arrestin-1 small interfering RNA, insulin-induced IRS-1 degradation was enhanced. Insulin stimulated the association of IRS-1 and Mdm2, an E3 ubiquitin ligase, and this association was inhibited to overexpression of WT beta-arrestin-1, which led by decreased ubiquitin content of IRS-1, suggesting that both beta-arrestin-1 and IRS-1 competitively bind to Mdm2. In summary, we have found the following: (i) beta-arrestin-1 can alter insulin signaling by inhibiting insulin-induced proteasomal degradation of IRS-1; (ii) beta-arrestin-1 decreases the rate of ubiquitination of IRS-1 by competitively binding to endogenous Mdm2, an E3 ligase that can ubiquitinate IRS-1; (iii) dephosphorylation of S412 on beta-arrestin and the amino terminus of beta-arrestin-1 are required for this effect of beta-arrestin on IRS-1 degradation; and (iv) inhibition of beta-arrestin-1 leads to enhanced IRS-1 degradation and accentuated cellular insulin resistance.

Acetylcysteine↗
PubMed2003

Multifaceted roles of beta-arrestins in the regulation of seven-membrane-spanning receptor trafficking and signalling.

Beta-arrestins are cytosolic proteins that bind to activated and phosphorylated G-protein-coupled receptors [7MSRs (seven-membrane-spanning receptors)] and uncouple them from G-protein-mediated second messenger signalling pathways. The binding of beta-arrestins to 7MSRs also leads to new signals via activation of MAPKs (mitogen-activated protein kinases) such as JNK3 (c-Jun N-terminal kinase 3), ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 MAPKs. By binding to endocytic proteins [clathrin, AP2 (adapter protein 2), NSF (N -ethylmaleimide-sensitive fusion protein) and ARF6 (ADP-ribosylation factor 6)], beta-arrestins also serve as adapters to link the receptors to the cellular trafficking machinery. Agonist-promoted ubiquitination of beta-arrestins is a prerequisite for their role in receptor internalization, as well as a determinant of the differing trafficking patterns of distinct classes of receptors. Recently, beta-arrestins have also been implicated as playing novel roles in cellular chemotaxis and apoptosis. By virtue of their ability to bind, in a stimulus-dependent fashion, to 7MSRs as well as to different classes of cellular proteins, beta-arrestins serve as versatile adapter proteins that regulate the signalling and trafficking of the receptors.

ADP-Ribosylation Factor 6↗
PubMed2003

Regulation of V2 vasopressin receptor degradation by agonist-promoted ubiquitination.

The seven-transmembrane-spanning vasopressin V2 receptor (V2R) is a Gs-coupled receptor that is rapidly phosphorylated and internalized following stimulation with the agonist, arginine-vasopressin. Herein, we show that the V2R is ubiquitinated following agonist stimulation. V2R-ubiquitination is not observed in a beta-arrestin1,2 deleted mouse fibroblast cell line and is restored following introduction of beta-arrestin2, thus indicating that beta-arrestin2 is required for the ubiquitination of V2R. A mutant V2R (K268R) that is not ubiquitinated still activates Gs and internalizes with similar kinetics as the wild type receptor. Unstimulated wild type and K268R mutant receptors degrade at similar rates and have comparable half-lives of 217 +/- 17 and 245 +/- 29 min as determined by pulse-chase experiments. However, following agonist stimulation, the rate of receptor degradation for the wild type is enhanced (half-life of 69 +/- 19 min), whereas that of the mutant is only minimally affected (half-life of 188 +/- 11 min). These data suggest that V2R levels are regulated through at least two processes. In the absence of agonist stimulation, a slow degradative pathway operates that is independent of receptor ubiquitination. However, receptor stimulation leads to rapid beta-arrestin2-dependent ubiquitination of the receptor and increased degradation.

Amino Acid Sequence↗
PubMed2003

Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2.

Stimulation of a mutant angiotensin type 1A receptor (DRY/AAY) with angiotensin II (Ang II) or of a wild-type receptor with an Ang II analog ([sarcosine1,Ile4,Ile8]Ang II) fails to activate classical heterotrimeric G protein signaling but does lead to recruitment of beta-arrestin 2-GFP and activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) (maximum stimulation approximately 50% of wild type). This G protein-independent activation of mitogen-activated protein kinase is abolished by depletion of cellular beta-arrestin 2 but is unaffected by the PKC inhibitor Ro-31-8425. In parallel, stimulation of the wild-type angiotensin type 1A receptor with Ang II robustly stimulates ERK1/2 activation with approximately 60% of the response blocked by the PKC inhibitor (G protein dependent) and the rest of the response blocked by depletion of cellular beta-arrestin 2 by small interfering RNA (beta-arrestin dependent). These findings imply the existence of independent G protein- and beta-arrestin 2-mediated pathways leading to ERK1/2 activation and the existence of distinct "active" conformations of a seven-membrane-spanning receptor coupled to each.

Amino Acid Sequence↗
PubMed2003

Trafficking patterns of beta-arrestin and G protein-coupled receptors determined by the kinetics of beta-arrestin deubiquitination.

Agonist-dependent internalization of G protein-coupled receptors via clathrin-coated pits is dependent on the adaptor protein beta-arrestin, which interacts with elements of the endocytic machinery such as AP2 and clathrin. For the beta(2)-adrenergic receptor (beta(2)AR) this requires ubiquitination of beta-arrestin by E3 ubiquitin ligase, Mdm2. Based on trafficking patterns and affinity of beta-arrestin, G protein-coupled receptors are categorized into two classes. For class A receptors (e.g. beta(2)AR), which recycle rapidly, beta-arrestin directs the receptors to clathrin-coated pits but does not internalize with them. For class B receptors (e.g. V2 vasopressin receptors), which recycle slowly, beta-arrestin internalizes with the receptor into endosomes. In COS-7 and human embryonic kidney (HEK)-293 cells, stimulation of the beta(2)AR or V2 vasopressin receptor leads, respectively, to transient or stable beta-arrestin ubiquitination. The time course of ubiquitination and deubiquitination of beta-arrestin correlates with its association with and dissociation from each type of receptor. Chimeric receptors, constructed by switching the cytoplasmic tails of the two classes of receptors (beta(2)AR and V2 vasopressin receptors), demonstrate reversal of the patterns of both beta-arrestin trafficking and beta-arrestin ubiquitination. To explore the functional consequences of beta-arrestin ubiquitination we constructed a yellow fluorescent protein-tagged beta-arrestin2-ubiquitin chimera that cannot be deubiquitinated by cellular deubiquitinases. This "permanently ubiquitinated" beta-arrestin did not dissociate from the beta(2)AR but rather internalized with it into endosomes, thus transforming this class A receptor into a class B receptor with respect to its trafficking pattern. Overexpression of this beta-arrestin ubiquitin chimera in HEK-293 cells also results in enhancement of beta(2)AR internalization and degradation. In the presence of N-ethylmaleimide (an inhibitor of deubiquitinating enzymes), coimmunoprecipitation of the receptor and beta-arrestin was increased dramatically, suggesting that deubiquitination of beta-arrestin triggers its dissociation from the receptor. Thus the ubiquitination status of beta-arrestin determines the stability of the receptor-beta-arrestin complex as well as the trafficking pattern of beta-arrestin.

Animals↗