Proteomic Analysis of the Epidermal Growth Factor Receptor (EGFR) Interactome and Post-translational Modifications Associated with Receptor Endocytosis in Response to EGF and Stress

Aberrant expression, activation, and stabilization of epidermal growth factor receptor (EGFR) are causally associated with several human cancers. Post-translational modifications and protein-protein interactions directly modulate the signaling and trafficking of the EGFR. Activated EGFR is internalized by endocytosis and then either recycled back to the cell surface or degraded in the lysosome. EGFR internalization and recycling also occur in response to stresses that activate p38 MAP kinase. Mass spectrometry was applied to comprehensively analyze the phosphorylation, ubiquitination, and protein-protein interactions of wild type and endocytosis-defective EGFR variants before and after internalization in response to EGF ligand and stress. Prior to internalization, EGF-stimulated EGFR accumulated ubiquitin at 7 K residues and phosphorylation at 7 Y sites and at S¹¹⁰⁴. Following internalization, these modifications diminished and there was an accumulation of S/T phosphorylations. EGFR internalization and many but not all of the EGF-induced S/T phosphorylations were also stimulated by anisomycin-induced cell stress, which was not associated with receptor ubiquitination or elevated Y phosphorylation. EGFR protein interactions were dramatically modulated by ligand, internalization, and stress. In response to EGF, different E3 ubiquitin ligases became maximally associated with EGFR before (CBL, HUWE1, and UBR4) or after (ITCH) internalization, whereas CBLB was distinctively most highly EGFR associated following anisomycin treatment. Adaptin subunits of AP-1 and AP-2 clathrin adaptor complexes also became EGFR associated in response to EGF and anisomycin stress. Mutations preventing EGFR phosphorylation at Y⁹⁹⁸ or in the S¹⁰³⁹ region abolished or greatly reduced EGFR interactions with AP-2 and AP-1, and impaired receptor trafficking. These results provide new insight into spatial, temporal, and mechanistic aspects of EGFR regulation.Receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR)¹ are aberrantly activated by mutation and/or over-expression in numerous human cancers (1, 2). Ligand-activated EGFR, similar to many receptor tyrosine kinases, is normally subject to clathrin-mediated endocytosis (CME) involving internalization and followed by sorting through the endosomal compartment (reviewed in 3). From endosomes, and as a function of which ligand is bound, the receptor may be recycled back to the cell surface or down-regulated as a consequence of trafficking to lysosomes for proteolytic degradation (4, 5). The EGFR also undergoes CME-mediated internalization and recycling back to the plasma membrane in response to cellular stresses that activate p38 MAPK, for example in response to the chemotherapeutic agent cisplatin, the antibiotic anisomycin, and the cytokine tumor necrosis factor-α (TNFα) (6–8). Various oncogenic mutations in the EGFR, as well as hetero-dimerization with other ErbB family members impairs EGFR down-regulation (9). This leads to aberrant, sustained EGFR signaling, which elicits cellular responses central to the cancer cell phenotype including cell proliferation, survival, motility/migration, and invasion (reviewed in 10).EGFR signaling and trafficking involve an overlapping set of factors that have been extensively reviewed (10, 11). These processes are products of EGFR protein-protein interactions and post-translational modifications (PTMs) including phosphorylation, ubiquitinylation, and lysine acetylation (12). Extracellular binding of ligand induces EGFR dimerization and trans-autophosphorylation at intracellular tyrosine residues, which serve as binding sites for various enzymes and adaptor proteins (11). These receptor-binding proteins are involved in signaling and/or receptor trafficking, and also lead to further modulation of receptor PTMs. For example, binding of the E3 ubiquitin ligase CBL at EGFR pY¹⁰⁶⁹ (13–15) or indirectly through the adaptor protein Grb2, which binds primarily at pY¹⁰⁹² (16), are both involved in EGFR ubiquitinylation and down-regulation (17). Although not an exclusive mechanism, EGFR internalization mainly involves clathrin and the AP-2 clathrin adaptor complex (12, 18–22) in addition to Grb2 (18, 23, 24). EGFR internalization and recycling in response to stress-induced p38 MAPK activation requires AP-2, but not Grb2 (18), and is reportedly independent of receptor kinase activity, tyrosine phosphorylation, and ubiquitination (6–8). Trafficking of endocytosed EGFR to the lysosome, but not the initial internalization step itself, requires CBL (25, 26), and is associated with ubiquitination at up to six lysine residues within the EGFR kinase domain (14). Additionally, ubiquitin-interacting endocytosis factors including Hrs, STAM, and STAM2 become tyrosine phosphorylated in response to EGFR activation (27), and EGFR ubiquitination is required for endosomal sorting (3). Gill and colleagues identified in the EGFR a region spanning residues 997–1046 as conferring endocytic function to otherwise endocytosis-defective EGF receptors truncated after the kinase domain (28). Consistent with this, EGFR phosphorylation sites linked with receptor trafficking are present within or proximal to this part of the receptor. For example, EGFR phosphorylation at S⁹⁹¹ and Y⁹⁹⁸ accumulate with relatively slow kinetics following stimulation of cells with EGF (29). Phosphorylation-defective variants Y998F and S991A are impaired for ligand-stimulated down-regulation relative to wild type (WT) EGFR, but remain proficient for rapid EGFR-to-ERK signaling (29). Non-phosphorylated Y⁹⁹⁸ was cited as part of an AP-2 binding motif (Y⁹⁹⁸RAL) (22), while a nearby di-leucine motif (LL^1034/35) also serves as an AP-2 binding site (22, 30). Phosphorylations at EGFR S¹⁰³⁹ and T¹⁰⁴¹ occur downstream of p38 MAPK in response to anisomycin-induced cell stress, and are also phosphorylated at lower levels as part of the normal cellular response to EGFR activation by EGF (29). The adaptor protein Odin (ANKS1A) becomes tyrosine phosphorylated prior to EGFR internalization following EGF treatment of cells, and functions as an effector of EGFR recycling (31). Therefore, in response to diverse extracellular signals a multitude of reversible PTMs and interacting proteins govern EGFR internalization, trafficking, and ultimately, stability and signaling. However, our understanding of spatial-temporal and mechanistic relationships of individual EGFR PTMs and protein interactions, and their biological consequences are largely qualitative and incomplete.The objective of the current study was to characterize and compare aspects of the initial, pre- and post-internalization stages of EGFR endocytosis in response to EGF and cell stress. A battery of methods was applied to identify and absolutely or relatively quantify EGFR phosphorylation, ubiquitination, and protein-protein interactions. These included fluorescence microscopic imaging, and quantitative LC-MS/MS including targeted measurements by selected reaction monitoring (SRM), and comprehensive quantification by using ultra high resolution MS. These were applied with an established model system based on human HEK293 cells engineered to express defined levels of wild type and various phosphorylation-defective EGFR variants tagged with the Flag epitope. The comprehensive analysis revealed distinctive patterns of EGFR modifications and interactions that correlated with receptor activation and internalization. Generally, EGF-stimulated EGFR tyrosine phosphorylations and lysine ubiquitinations, which were maximal prior to internalization, decreased 15-min after receptor internalization was initiated, whereas S/T phosphorylations increased. A subset of EGF-stimulated S/T phosphorylations including pS⁹⁹¹ and pS¹⁰³⁹ and proximal S/T residues accumulated to an even greater extent in response to anisomycin. EGFR variants with amino acid substitutions at these positions were largely impaired for AP-1 and AP-2 interactions, showed altered patterns of ubiquitination, and resistance to EGF-stimulated receptor down-regulation. These results provide new insight into the dynamics and molecular events associated with EGFR function.     

Epidermal Growth Factor Receptor Phosphorylation Sites Ser991 and Tyr998 Are Implicated in the Regulation of Receptor Endocytosis and Phosphorylations at Ser1039 and Thr1041

Aberrant expression, activation, and down-regulation of the epidermal growth factor receptor (EGFR) have causal roles in many human cancers, and post-translational modifications including phosphorylation and ubiquitination and protein-protein interactions directly modulate EGFR function. Quantitative mass spectrometric analyses including selected reaction monitoring (also known as multiple reaction monitoring) were applied to the EGFR and associated proteins. In response to epidermal growth factor (EGF) stimulation of cells, phosphorylations at EGFR Ser⁹⁹¹ and Tyr⁹⁹⁸ accumulated more slowly than at receptor sites involved in RAS-ERK signaling. Phosphorylation-deficient mutant receptors S991A and Y998F activated ERK in response to EGF but were impaired for receptor endocytosis. Consistent with these results, the mutant receptors retained a network of interactions with known signaling proteins including EGF-stimulated binding to the adaptor GRB2. Compared with wild type EGFR the Y998F variant had diminished EGF-stimulated interaction with the ubiquitin E3 ligase CBL, and the S991A variant had decreased associated ubiquitin. The endocytosis-defective mutant receptors were found to have elevated phosphorylation at positions Ser¹⁰³⁹ and Thr¹⁰⁴¹. These residues reside in a serine/threonine-rich region of the receptor previously implicated in p38 mitogen-activated protein kinase-dependent stress/cytokine-induced EGFR internalization and recycling (Zwang, Y., and Yarden, Y. (2006) p38 MAP kinase mediates stress-induced internalization of EGFR: implications for cancer chemotherapy. EMBO J. 25, 4195–4206). EGF-induced phosphorylations at Ser¹⁰³⁹ and Thr¹⁰⁴¹ were blocked by treatment of cells with SB-202190, a selective inhibitor of p38. These results suggest that coordinated phosphorylation of EGFR involving sites Tyr⁹⁹⁸, Ser⁹⁹¹, Ser¹⁰³⁹, and Thr¹⁰⁴¹ governs the trafficking of EGF receptors. This reinforces the notion that EGFR function is manifest through spatially and temporally controlled protein-protein interactions and phosphorylations.Upon activation by ligand, the epidermal growth factor receptor (EGFR)¹ dimerizes, sometimes as heterodimers with other EGFR family members; is catalytically activated by reorientation of kinase region subdomains; becomes covalently modified by phosphorylation and ubiquitination; and interacts with a variety of intracellular proteins (1, 2). These events activate intracellular signaling cascades, and concurrently the dimerized receptors become internalized through endocytosis and then may be recycled to the cell surface or degraded in lysosomes (3). Systematic analysis of EGFR family phosphorylation-dependent protein interactions has been assessed (4, 5), and many of the known EGFR-interacting proteins can be categorized as functioning in cellular processes such as EGF-induced signal transduction and EGFR endocytosis and trafficking. Temporal analysis of tyrosine phosphorylation following EGF treatment of cells revealed groups of EGFR substrates with shared profiles of phosphorylation kinetics, including some that display rapid kinetics of phosphorylation accumulation and are involved in signal transduction (e.g. ERK kinase activation) and others that accumulate more slowly following ligand treatment and are involved in receptor internalization and down-regulation (5–11). Although advances in MS and the definition of phosphorylation-dependent protein-protein interactions have led to a greatly expanded view of EGFR function and regulation, our understanding of the biological consequences and spatial-temporal relationships of individual modifications is incomplete.In a previous quantitative phosphoproteomics study aimed at the identification of drug-modulated changes in phosphorylation associated with the EGFR network, a cluster of three sites of phosphorylation in the EGFR carboxyl tail region was identified as affected by receptor stimulation by EGF and inhibited by the ATP-competitive EGFR inhibitor PKI166 in human A431 tumor cells and xenograft tumors (12). The three sites in the cluster, Ser⁹⁹¹,² Ser⁹⁹⁵, and Tyr⁹⁹⁸, are localized within a single tryptic peptide having the sequence MHLPSPTDSNFYR that spans residues 987–999. The phosphorylation of Tyr⁹⁹⁸ was first described by Stover et al. (12), whereas the two serine sites were shown previously to be phosphorylated by Heisermann and Gill (13). Numerous recent studies using different cultured cell models have verified the phosphorylation of EGFR at Tyr⁹⁹⁸ and Ser⁹⁹¹ (10, 11, 14, 15), and Thr⁹⁹³ was also observed to be phosphorylated within this same region of the EGFR in EGF-stimulated HeLa cells (10). The modulation of these sites by EGF and the EGFR inhibitor implicates them in EGFR signaling and suggests that they may have utility as pharmacodynamic markers of EGFR activity. However, the function and importance of these sites, their modulation by kinases and phosphatases, and possible roles in EGFR function remain unknown.Several amino acid residues in the EGFR have been implicated in the regulation of its trafficking. Sorkin et al. (16) showed that substitution of Tyr⁹⁹⁸ with phenylalanine rendered high density EGFRs defective for endocytosis and interaction with AP-2. More recent kinetic studies using MS indicated that EGFR phosphorylation at both Tyr⁹⁹⁸ (5) and Ser⁹⁹¹ (10) occurs relatively slowly compared with other EGF-induced tyrosine phosphorylations known to be involved in receptor-proximal signal transduction. For example, Mann and co-workers (10) recorded maximal phosphorylation at EGFR sites Tyr¹¹¹⁰, Tyr¹¹⁷², and Tyr¹¹⁹⁷ at 1 min post-EGF, whereas EGF-stimulated phosphorylation at Tyr⁹⁹⁸ was still increasing at 15 min post-EGF (5), and a peptide containing both Ser(P)⁹⁹¹ and Thr(P)⁹⁹³ peaked after 10 min (10). However, the role of phosphorylation at Tyr⁹⁹⁸ and Ser⁹⁹¹ has not been reported. Another region of the EGFR, spanning residues 1026–1046, was identified by Zwang and Yarden (17) as a target of phosphorylation downstream of the stress-activated mitogen-activated protein (MAP) kinase p38 and associated with transient internalization and recycling of the EGFR in response to cytokine (TNFα) and stress challenges such as UV irradiation and the chemotherapeutic agent cisplatinum. Within this part of the receptor, a 13-residue section spanning 1029–1041 and the leucines at 1034 and 1035 in particular were found to be essential for ligand- and dimerization-induced EGFR endocytosis (18). Although both EGF- and stress-induced EGFR internalization may be clathrin-mediated, they differ in that the former leads to receptor down-regulation and involves the E3 ubiquitin ligase CBL (19), whereas the latter involves receptor recycling, is not associated with receptor phosphorylation at the CBL binding site Tyr(P)¹⁰⁶⁹, and, in the case of TNFα treatment, involves activation of the transforming growth factor β-activated kinase TAK1 upstream of p38 (20). Interestingly although p38 kinase is not required for EGF-induced EGFR internalization, it is required for CBL-dependent receptor degradation (21). Therefore, alternate pathways involving p38 kinase regulate the down-regulation or recycling of the EGFR in response to diverse extracellular signals. However, the molecular details that govern these two processes are not fully understood.In the current study, EGFR phosphorylation, signaling, protein-protein interactions, and trafficking were analyzed to address the role of Tyr⁹⁹⁸ and Ser⁹⁹¹ in EGFR endocytosis. This was achieved by application of complementary methods including quantitative selected reaction monitoring (SRM, also known as MRM for multiple reaction monitoring) mass spectrometry, fluorescence imaging and cell sorting, immunoaffinity protein enrichment and blotting, and site-directed mutagenesis. Substitution mutations that prevented phosphorylation at EGFR Tyr⁹⁹⁸ and Ser⁹⁹¹ did not prevent EGFR-to-ERK signaling but impaired EGF-induced receptor internalization and stimulated p38 kinase-dependent receptor phosphorylation at positions Ser¹⁰³⁹ and Thr¹⁰⁴¹. These findings confirm the importance of Tyr⁹⁹⁸ and reveal a role for Ser⁹⁹¹ in EGF-mediated EGFR internalization possibly involving cross-talk with the p38 kinase-dependent EGFR recycling pathway.     

Bile Acid-induced Epidermal Growth Factor Receptor Activation in Quiescent Rat Hepatic Stellate Cells Can Trigger Both Proliferation and Apoptosis

Bile acids have been reported to induce epidermal growth factor receptor (EGFR) activation and subsequent proliferation of activated hepatic stellate cells (HSC), but the underlying mechanisms and whether quiescent HSC are also a target for bile acid-induced proliferation or apoptosis remained unclear. Therefore, primary rat HSC were cultured for up to 48 h and analyzed for their proliferative/apoptotic responses toward bile acids. Hydrophobic bile acids, i.e. taurolithocholate 3-sulfate, taurochenodeoxycholate, and glycochenodeoxycholate, but not taurocholate or tauroursodeoxycholate, induced Yes-dependent EGFR phosphorylation. Simultaneously, hydrophobic bile acids induced phosphorylation of the NADPH oxidase subunit p47^phox and formation of reactive oxygen species (ROS). ROS production was sensitive to inhibition of acidic sphingomyelinase, protein kinase Cζ, and NADPH oxidases. All maneuvers which prevented bile acid-induced ROS formation also prevented Yes and subsequent EGFR phosphorylation. Taurolithocholate 3-sulfate-induced EGFR activation was followed by extracellular signal-regulated kinase 1/2, but not c-Jun N-terminal kinase (JNK) activation, and stimulated HSC proliferation. When, however, a JNK signal was induced by coadministration of cycloheximide or hydrogen peroxide (H₂O₂), activated EGFR associated with CD95 and triggered EGFR-mediated CD95-tyrosine phosphorylation and subsequent formation of the death-inducing signaling complex. In conclusion, hydrophobic bile acids lead to a NADPH oxidase-driven ROS generation followed by a Yes-mediated EGFR activation in quiescent primary rat HSC. This proliferative signal shifts to an apoptotic signal when a JNK signal simultaneously comes into play.Hydrophobic bile acids play a major role in the pathogenesis of cholestatic liver disease and are potent inducers of hepatocyte apoptosis by triggering a ligand-independent activation of the CD95² death receptor (1–5). The underlying molecular mechanisms are complex and involve a Yes-dependent, but ligand-independent activation of the epidermal growth factor receptor (EGFR), which catalyzes CD95-tyrosine phosphorylation as a prerequisite for CD95 oligomerization, formation of the death-inducing signaling complex (DISC), and apoptosis induction (6, 7). Bile acids also activate EGFR in cholangiocytes (8) and activated hepatic stellate cells (HSC) (9), however, the mechanisms underlying bile acid-induced EGFR activation in HSC remained unclear (9). Surprisingly, bile acid-induced EGFR activation in HSC does not trigger apoptosis but results in a stimulation of cell proliferation (9). The behavior of quiescent HSC toward CD95 ligand (CD95L) is also unusual. CD95L, which is a potent inducer of hepatocyte apoptosis (10–12), triggers activation of the EGFR in quiescent HSC, stimulates HSC proliferation, and simultaneously inhibits CD95-dependent death signaling through CD95-tyrosine nitration (13). Similar observations were made with other death receptor ligands, i.e. tumor necrosis factor-α (TNF-α) and TNF-related apoptosis-inducing ligand (TRAIL) (13). The mitogenic action of CD95L in quiescent, 1–2-day cultured HSC is because of a c-Src-dependent shedding of EGF and subsequent auto/paracrine activation of the EGFR (13). This unusual behavior of quiescent HSC toward death receptor ligands may relate to the recent findings that quiescent HSC might represent a stem/progenitor cell compartment in the liver with a capacity to differentiate not only into myofibroblasts but also toward hepatocyte- and endothelial-like cells (14). Thus, stimulation of HSC proliferation and resistance toward apoptosis in the hostile cytokine milieu accompanying liver injury may help HSC to play their role in liver regeneration. During cholestatic liver injury quiescent HSC are exposed to increased concentrations of circulating bile acids, but it is not known whether this may lead to HSC proliferation (as shown for activated HSC) (9), HSC apoptosis (as shown for hepatocytes) (1–7), or both of them. Therefore, the aim of the current study was (a) to identify the molecular mechanisms underlying bile acid-induced EGFR activation and (b) to elucidate whether bile acid-induced signaling can couple to both cell proliferation and cell death in quiescent HSC.The present study shows that cholestatic bile acids trigger a rapid NADPH oxidase activation in quiescent HSC, which leads to a Yes-mediated EGFR phosphorylation and HSC proliferation. In contrast to hepatocytes, hydrophobic bile acids do not induce a JNK signal in HSC. However, when JNK activation is induced by coadministration of either cycloheximide (CHX) or hydrogen peroxide (H₂O₂), the bile acid-induced mitogenic signal is shifted to an apoptotic one.     

Phosphorylation-independent Regulation of Metabotropic Glutamate Receptor 5 Desensitization and Internalization by G Protein-coupled Receptor Kinase 2 in Neurons

The uncoupling of metabotropic glutamate receptors (mGluRs) from heterotrimeric G proteins represents an essential feedback mechanism that protects neurons against receptor overstimulation that may ultimately result in damage. The desensitization of mGluR signaling is mediated by both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). Unlike mGluR1, the attenuation of mGluR5 signaling in HEK 293 cells is reported to be mediated by a phosphorylation-dependent mechanism. However, the mechanisms regulating mGluR5 signaling and endocytosis in neurons have not been investigated. Here we show that a 2-fold overexpression of GRK2 leads to the attenuation of endogenous mGluR5-mediated inositol phosphate (InsP) formation in striatal neurons and siRNA knockdown of GRK2 expression leads to enhanced mGluR5-mediated InsP formation. Expression of a catalytically inactive GRK2-K220R mutant also effectively attenuates mGluR5 signaling, but the expression of a GRK2-D110A mutant devoid in Gα_q/11 binding increases mGluR5 signaling in response to agonist stimulation. Taken together, these results indicate that the attenuation of mGluR5 responses in striatal neurons is phosphorylation-independent. In addition, we find that mGluR5 does not internalize in response to agonist treatment in striatal neuron, but is efficiently internalized in cortical neurons that have higher levels of endogenous GRK2 protein expression. When overexpressed in striatal neurons, GRK2 promotes agonist-stimulated mGluR5 internalization. Moreover, GRK2-mediated promotion of mGluR5 endocytosis does not require GRK2 catalytic activity. Thus, we provide evidence that GRK2 mediates phosphorylation-independent mGluR5 desensitization and internalization in neurons.Glutamate is the major excitatory neurotransmitter in the mammalian brain and functions to activate two distinct classes of receptors (ionotropic and metabotropic) to regulate a variety of physiological functions (1–3). Ionotropic glutamate receptors, such as NMDA, AMPA, and kainate receptors, are ligand-gated ion channels, whereas metabotropic glutamate receptors (mGluRs)⁵ are members of the G protein-coupled receptor (GPCR) superfamily (4–7). mGluRs modulate synaptic activity via the activation of heterotrimeric G proteins that are coupled to a variety of second messenger cascades. Group I mGluRs (mGluR1 and mGluR5) are coupled to the activation of Gα_q/11 proteins, which stimulate the activation of phospholipase Cβ1 resulting in diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP₃) formation, release of Ca²⁺ from intracellular stores and subsequent activation of protein kinase C.The attenuation of GPCR signaling is mediated in part by G protein-coupled receptor kinases (GRKs), which phosphorylate GPCRs to promote the binding of β-arrestin proteins that uncouple GPCRs from heterotrimeric G proteins (8–10). GRK2 has been demonstrated to contribute to the phosphorylation and desensitization of both mGluR1 and mGluR5 in human embryonic kidney (HEK 293) cells (11–17). GRK4 is also implicated in mediating the desensitization of mGluR1 signaling in cerebellar Purkinje cells, but does not contribute to the desensitization of mGluR5 (14, 15). In addition, GRK4 plays a major role in mGluR1 internalization (13, 14). A role for GRK2 in promoting mGluR1 internalization is less clear as different laboratories have obtained discordant results (11, 14, 15, 16). However, the only study examining the role of GRK2 in regulating mGluR1 endocytosis in a native system reported that GRK2 knockdown had no effect upon mGluR1 internalization in cerebellar Purkinje cells (14).GRK2 is composed of three functional domains: an N-terminal regulator of G protein signaling (RGS) homology (RH) domain, a central catalytic domain, and a C-terminal G_βγ binding pleckstrin homology domain (18). In HEK 293 cells, mGluR1 desensitization is not dependent on GRK2 catalytic activity. Rather the GRK2 RH domain interacts with both the second intracellular loop domain of mGluR1 and the α-subunit of Gα_q/11 and attenuates second messenger responses by disrupting the mGluR1/Gα_q/11 signaling complexes (12, 19–21). Although the molecular mechanism underlying GRK2-mediated attenuation of mGluR1 signaling is relatively well established in HEK 293 cells, the role of GRK2 in regulating the desensitization of mGluRs in neurons remains to be determined. Moreover, it is not known whether GRK2-dependent attenuation of mGluR5 signaling is mediated by the same phosphorylation-independent mechanism that has been described for mGluR1. In a previous study, GRK2-mediated mGluR5 desensitization was reported to be phosphorylation-dependent, based on the observation that the overexpression of a catalytically inactive GRK2 (K220R) did not attenuate mGluR5 signaling (15). In the present study, we examined whether a 2-fold overexpression of GRK2 in primary mouse striatal neurons to match GRK2 expression levels found in the cortex results in increased agonist-stimulated desensitization and internalization of endogenous mGluR5. We report here that GRK2 mediates phosphorylation-independent mGluR5 desensitization and internalization. Furthermore, GRK2 knockdown causes an increase in mGluR5 signaling, demonstrating that endogenous GRK2 plays a role in mGluR5 desensitization.     

Cell Proliferation and Epidermal Growth Factor Signaling in Non-small Cell Lung Adenocarcinoma Cell Lines Are Dependent on Rin1

Rin1 is a Rab5 guanine nucleotide exchange factor that plays an important role in Ras-activated endocytosis and growth factor receptor trafficking in fibroblasts. In this study, we show that Rin1 is expressed at high levels in a large number of non-small cell lung adenocarcinoma cell lines, including Hop62, H650, HCC4006, HCC827, EKVX, HCC2935, and A549. Rin1 depletion from A549 cells resulted in a decrease in cell proliferation that was correlated to a decrease in epidermal growth factor receptor (EGFR) signaling. Expression of wild type Rin1 but not the Rab5 guanine nucleotide exchange factor-deficient Rin1 (Rin1Δ) complemented the Rin1 depletion effects, and overexpression of Rin1Δ had a dominant negative effect on cell proliferation. Rin1 depletion stabilized the cell surface levels of EGFR, suggesting that internalization was necessary for robust signaling in A549 cells. In support of this conclusion, introduction of either dominant negative Rab5 or dominant negative dynamin decreased A549 proliferation and EGFR signaling. These data demonstrate that proper internalization and endocytic trafficking are critical for EGFR-mediated signaling in A549 cells and suggest that up-regulation of Rin1 in A549 cell lines may contribute to their proliferative nature.Internalization of epidermal growth factor receptors (EGFR)² and their subsequent delivery to lysosomes play key roles in attenuating EGF-mediated signaling cascades (1, 2). The proper delivery of EGFR into lysosomes for degradation requires a series of highly regulated targeting and delivery events. Following ligand binding, EGFR is internalized via endocytic vesicles that are subsequently targeted to early endosomes. This targeting event is mediated by the small GTPase, Rab5 (3, 4). Once delivered to the early endosome, receptors that are destined for degradation are incorporated into vesicles that bud into the lumen of the endosome, forming the multivesicular body (reviewed in Refs. 5, 6). Sequestration of the activated cytoplasmic domain of EGFR into the intralumenal vesicles of the multivesicular body effectively terminates receptor signaling (7). Subsequent fusion of the multivesicular body with lysosomes delivers the intralumenal vesicles and their contents into the lumen of the lysosome where they are degraded (reviewed in Refs. 8–10). Inactivating mutations in Rab5 disrupt the delivery of cell surface receptors, such as EGFR, to early endosomes, thereby inhibiting receptor trafficking to the lysosome and receptor degradation (11, 12). Therefore, activation of Rab5 is a key point of regulation for EGFR signaling.Rab5 cycles between an inactive GDP-bound state and an active GTP-bound state, and Rab5 activation requires the exchange of GDP to GTP. This exchange is catalyzed by guanine nucleotide exchange factors (GEFs) that are specific to the Rab5 family of proteins (reviewed in Ref. 13). Rab5 family GEFs all contain a catalytic vacuolar protein sorting 9 (Vps9) domain that facilitates the GDP to GTP exchange (14–17). Many Rab5 GEFs contain other functional domains that are involved in cell signaling events (13). Rin1 is a good example of a multidomain Rab5 GEF. In addition to the Vps9 domain, Rin1 also contains an Src homology 2 domain, a proline-rich domain, and a Ras association domain. Rin1 was originally identified through its ability to interact with active Ras (18), and a role for Rin1 in a number of cell signaling systems has been established, including EGF-mediated signaling (19–21). Rin1 directly interacts with the activated EGFR through its Src homology 2 domain (22). Furthermore, Ras occupation of the Rin1 Ras association domain positively impacts the Rab5 GEF activity of Rin1, which promotes EGFR internalization and attenuation in fibroblasts (23). However, Rin1 expression is up-regulated in several types of cancers, including squamous cell carcinoma (24), colorectal cancer (25), and cervical cancer (26), through duplications or rearrangements of the RIN1 locus. These studies suggest that Rin1 may also play a role in enhancing cell proliferation.It is well established that a large percentage of non-small cell lung adenocarcinomas exhibit up-regulation of EGFR and aberrant signaling through the Ras/MAPK pathway (reviewed in Ref. 27). In addition, a recent study examining 188 human lung adenocarcinomas identified that 132 of 188 tumor samples exhibited mutations relating to the Ras/MAPK signaling pathway (28). Accordingly, the role of Rin1 in non-small cell lung adenocarcinoma was addressed. Examination of a panel of non-small cell lung adenocarcinoma lines (including A549) revealed enhanced Rin1 expression relative to a nontransformed lung epithelial cell line (BEAS-2B). Depletion of Rin1 from A549 cells resulted in decreased proliferation. This decrease correlated with a reduction in EGF-activated ERK phosphorylation and the stabilization of cell surface EGFR. These defects were complemented by wild type Rin1 expression but not by mutant Rin1 lacking a functional Vps9 domain, suggesting that the GEF activity of Rin1 is necessary for proper EGFR signaling in A549 cells. In addition, overexpression of Rin1Δ, dominant negative Rab5, and dynamin resulted in similar defects in cell proliferation and EGFR signaling as Rin1 depletion. These data indicate that proper EGFR internalization and trafficking are critical for robust EGFR-mediated signaling and cell proliferation in A549 cells and offer evidence that Rin1 positively regulates cell proliferation in non-small cell lung adenocarcinoma.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]