AXL is a potential therapeutic target in dedifferentiated and pleomorphic liposarcomas.

BACKGROUND: AXL is a well-characterized, protumorigenic receptor tyrosine kinase that is highly expressed and activated in numerous human carcinomas and sarcomas, including aggressive subtypes of liposarcoma. However, the role of AXL in the pathogenesis of well-differentiated (WDLPS), dedifferentiated (DDLPS), and pleomorphic liposarcoma (PLS) has not yet been determined. METHODS: Immunohistochemical analysis of AXL expression was conducted on two tissue microarrays containing patient WDLPS, DDLPS, and PLS samples. A panel of DDLPS and PLS cell lines were interrogated via western blot for AXL expression and activity and by ELISA for growth arrest-specific 6 (GAS6) production. AXL knockdown was achieved by siRNA or shRNA. The effects of AXL knockdown on cell proliferation, migration, and invasion were measured in vitro. In addition, AXL shRNA-containing DDLPS cells were assessed for their tumor-forming capacity in vivo. RESULTS: In this study, we determined that AXL is expressed in a subset of WDLPS, DDLPS, and PLS patient tumor samples. In addition, AXL and its ligand GAS6 are expressed in a panel of DDLPS and PLS cell lines. We show that the in vitro activation of AXL via stimulation with exogenous GAS6 resulted in a significant increase in cell proliferation, migration, and invasion in DDLPS and PLS cell lines. Transient knockdown of AXL resulted in attenuation of these protumorigenic phenotypes in vitro. Stable AXL knockdown not only decreased migratory and invasive characteristics of DDLPS and PLS cells in vitro but also significantly diminished tumorigenicity of two dedifferentiated liposarcoma xenograft models in vivo. CONCLUSIONS: Our results suggest that AXL signaling contributes to the aggressiveness of DDLPS and PLS, and that AXL is therefore a potential therapeutic target for treatment of these rare, yet devastating tumors.

Naturally evolved G protein-coupled receptors adopt metastable conformations.

A wide range of membrane receptors signal through conformational changes, and the resulting protein conformational flexibility often hinders their structural studies. Because the determinants of membrane receptor conformational stability are still poorly understood, identifying a minimal set of perturbations stabilizing a membrane protein in a given conformation remains a major challenge in membrane protein structure determination. We present a novel approach integrating bioinformatics, computational design and experimental techniques that identifies and stabilizes metastable receptor regions. When applied to the beta1-adrenergic receptor, the method generated 13 novel receptor variants stabilized in the intended inactive state among which two exhibit an apparent thermostability higher than WT and M23 (a receptor variant previously stabilized by extensive scanning mutagenesis) by more than 30 °C and 11 °C, respectively. Targeted regions involve nonconserved unsatisfied polar residues or exhibit significant packing defects, features found in all class A G protein-coupled receptor structures. These findings suggest that natural G protein-coupled receptor sequences have evolved to be conformationally metastable through the design of suboptimal polar and van der Waals tertiary interactions. Given sufficiently accurate structural models, our approach should prove useful for designing stabilized variants of many uncharacterized membrane receptors.

Glucagon-like peptide-2-stimulated protein synthesis through the PI 3-kinase-dependent Akt-mTOR signaling pathway.

Glucagon-like peptide-2 (GLP-2) is a nutrient-responsive neuropeptide that exerts diverse actions in the gastrointestinal tract, including enhancing mucosal cell survival and proliferation. GLP-2 stimulates mucosal growth in vivo with an increased rate of protein synthesis. However, it was unclear whether GLP-2 can directly stimulate protein synthesis. The objective was to test critically whether GLP-2 receptor (GLP-2R) activation directly stimulates protein synthesis through a PI 3-kinase-dependent Akt-mTOR signaling pathway. HEK 293 cells (transfected with human GLP-2R cDNA) were treated with human GLP-2 with/without pretreatment of PI 3-kinase inhibitor (LY-294002) or mTOR inhibitor (rapamycin). Results show that 1) GLP-2 specifically bound to GLP-2R overexpressed in the HEK cells with K(a) = 0.22 nM and B(max) = 321 fmol/µg protein; 2) GLP-2-stimulated protein synthesis was dependent on the amount of GLP-2R cDNA and the dosage of GLP-2 and reached the plateau among 0.2-2 nM GLP-2; 3) GLP-2-stimulated protein synthesis was abolished by the PI 3-kinase inhibitor and mTOR inhibitor; and 4) GLP-2-mediated stimulation of phosphorylation on Akt and mTOR was dependent on the amount of GLP-2R cDNA transfected and the dosage of GLP-2. In addition, GLP-2-mediated action and signaling in regulation of protein synthesis were confirmed in mouse hippocampal neurons (expressing native GLP-2R). GLP-2 directly stimulated protein synthesis of primary cultured neurons in dosage-dependent, PI 3-kinase-dependent, and rapamycin-sensitive manners, which linked with activation of Akt-mTOR signaling pathway as well. We conclude that GLP-2R activation directly stimulates protein synthesis by activating the PI 3-kinase-dependent Akt-mTOR signaling pathway. GLP-2-stimulated protein synthesis may be physiologically relevant to maintaining neuronal long-term potentiation and providing secondary mediators (namely neuropeptides or growth factors).

ADP-ribosylation factors modulate the cell surface transport of G protein-coupled receptors.

ADP-ribosylation factors (ARFs) regulate vesicular traffic through recruiting coat proteins. However, their functions in the anterograde transport of nascent G protein-coupled receptors (GPCRs) from the endoplasmic reticulum to the plasma membrane remain poorly explored. Here we show that treatment with brefeldin A, an inhibitor of guanine nucleotide exchange on ARFs, markedly attenuated the cell surface numbers of alpha(2B)-adrenergic receptor (AR), beta(2)-AR, angiotensin II type 1 receptor, and chemokine (CXC motif) receptor 4. Functional inhibition of individual ARF GTPases by transient expression of the GDP-bound, GTP-bound, and guanine nucleotide-deficient mutants showed that the five human ARFs differentially modulated receptor cell surface expression and that the ARF1 mutants produced the most profound inhibitory effect. Furthermore, expression of the ARF1 GTPase-activating protein (GAP) ARFGAP1 significantly blocked receptor transport. Interestingly, the GDP- and GTP-bound ARF1 mutants arrested the receptors in distinct intracellular compartments. Consistent with the reduced receptor cell surface expression, extracellular signal-regulated kinase 1 and 2 activation by receptor agonists was significantly attenuated by the GDP-bound mutant ARF1T31N. Moreover, coimmunoprecipitation showed that alpha(2B)-AR associated with ARF1 and glutathione transferase pull-down assay indicated that the alpha(2B)-AR C terminus directly interacted with ARF1. These data show that ARF1 GTPase is involved in the regulation of cell surface expression of GPCRs at multiple transport steps.

Anterograde trafficking of G protein-coupled receptors: function of the C-terminal F(X)6LL motif in export from the endoplasmic reticulum.

We have reported previously that the F(X)(6)LL motif in the C termini is essential for export of alpha(2B)-adrenergic (alpha(2B)-AR) and angiotensin II type 1 receptors (AT1Rs) from the endoplasmic reticulum (ER). Here, we further demonstrate that mutation of the F(X)(6)LL motif similarly abolished the cell-surface expression of alpha(2B)-AR, AT1R, alpha(1B)-AR, and beta(2)-AR, suggesting that the F(X)(6)LL motif plays a general role in ER export of G protein-coupled receptors (GPCRs). Mutation of Phe to Val, Leu, Trp, and Tyr, and mutation of LL to FF and VV, markedly inhibited alpha(2B)-AR transport, indicating that the F(X)(6)LL function cannot be fully substituted by other hydrophobic residues. The structural analysis revealed that the Phe residue in the F(X)(6)LL motif is buried in the transmembrane domains and possibly interacts with Ile58 in beta(2)-AR and Val42 in alpha(2B)-AR, whereas the LL motif is exposed to the cytosolic space. Indeed, mutation of Ile58 in beta(2)-AR and Val42 in alpha(2B)-AR markedly disrupted cell surface transport of the receptors. It is noteworthy that the Val and Ile residues are highly conserved among the GPCRs carrying the F(X)(6)LL motif. Furthermore, the Phe mutant exhibited a stronger interaction with ER chaperones and was more potently rescued by physical and chemical treatments than the LL mutant. These data suggest that the Phe residue is probably involved in folding of alpha(2B)-AR and beta(2)-AR, possibly through interaction with other hydrophobic residues in neighboring domains. These data also provide the first evidence implying crucial roles of the C termini possibly through modulating multiple events in anterograde trafficking of GPCRs.

Endoplasmic reticulum export of adrenergic and angiotensin II receptors is differentially regulated by Sar1 GTPase.

The molecular mechanism underlying the export of G protein-coupled receptors (GPCRs) from the endoplasmic reticulum (ER) remains largely unknown. In this manuscript, we investigated the role of Sar1 GTPase, which coordinates the assembly and budding of COPII-coated vesicles, in the cell-surface targeting, signaling and ER export of alpha(2B)-adrenergic (alpha(2B)-AR), beta(2)-AR and angiotensin II type 1 receptors (AT1R). The cell-surface expression of alpha(2B)-AR, beta(2)-AR and AT1R, and receptor-mediated ERK1/2 activation were significantly attenuated by the GTP-bound mutant Sar1H79G, suggesting that export from the ER of these receptors is mediated through the Sar1-dependent COPII-coated vesicles. Interestingly, subcellular distribution analyses showed that alpha(2B)-AR and AT1R were highly concentrated at discrete locations near the nucleus in cells expressing Sar1H79G, whereas beta(2)-AR exhibited an ER distribution. These data indicate that Sar1-catalyzed efficient GTP hydrolysis differentially regulates ER export of adrenergic and angiotensin II receptors. These data provide the first evidence indicating distinct mechanisms for the recruitment of different GPCRs into the COPII vesicles on the ER membrane.

Analysis of Rab1 function in cardiomyocyte growth.

Protein transport between intracellular organelles is coordinated by Rab GTPases. As an initial approach to defining the function of Rab GTPases in cardiomyocytes, our laboratory focused on Rab1, which regulates protein transport specifically from the endoplasmic reticulum (ER) to the Golgi apparatus. Our studies have demonstrated that adenovirus-driven expression of Rab1 promotes cell growth of primary cultures of neonatal cardiomyocytes in vitro and that transgenic expression of Rab1 in the myocardium induces cardiac hypertrophy in mouse hearts in vivo. These data provide strong evidence implicating that ER-to-Golgi protein transport functions as a regulatory site for control of cardiomyocyte growth. Here we describe a sets of methods used in our laboratory to characterize the function of Rab1 GTPase in modulating cardiac myocyte growth.

Cell-surface targeting of alpha2-adrenergic receptors -- inhibition by a transport deficient mutant through dimerization.

We previously demonstrated that the alpha2B-adrenergic receptor mutant, in which the F(x)6IL motif in the membrane-proximal carboxyl terminus were mutated to alanines (alpha2B-ARm), is deficient in export from the endoplasmic reticulum (ER). In this report, we determined if alpha2B-ARm could modulate transport from the ER to the cell surface and signaling of its wild-type counterpart. Transient expression of alpha2B-ARm in HEK293T cells markedly inhibited cell-surface expression of wild-type alpha2B-AR, as measured by radioligand binding. Subcellular localization demonstrated that alpha2B-ARm trapped alpha2B-AR in the ER. The alpha2B-AR was shown to form homodimers and heterodimers with alpha2B-ARm as measured by co-immunoprecipitation of the receptors tagged with green fluorescent protein and hemagglutinin epitopes. In addition to alpha2B-AR, the transport of alpha2A-AR and alpha2C-AR to the cell surface was also inhibited by alpha2B-ARm. Furthermore, transient expression of alpha2B-ARm significantly reduced cell-surface expression of endogenous alpha2-AR in NG108-15 and HT29 cells. Consistent with its effect on alpha2-AR cell-surface expression, alpha2B-ARm attenuated alpha2A-AR- and alpha2B-AR-mediated ERK1/2 activation. These data demonstrated that the ER-retained mutant alpha2B-ARm conferred a dominant negative effect on the cell-surface expression of wild-type alpha2-AR, which is likely mediated through heterodimerization. These data indicate a crucial role of ER export in the regulation of cell-surface targeting and signaling of G protein-coupled receptors.

The mechanism and function of mitogen-activated protein kinase activation by ARF1.

Mitogen-activated protein kinases (MAPK) can be activated by a number of biochemical pathways through distinct signaling molecules. We have recently revealed a novel function for the Ras-like small GTPase ADP-ribosylation factor 1 (ARF1) in mediating the activation of Raf1-MEK-ERK1/2 pathway by G protein-coupled receptors [Dong C, Li C and Wu G (2011) J Biol Chem 286, 43,361-43,369]. Here, we have further defined the underlying mechanism and the possible function of ARF1-mediated MAPK pathway. We demonstrated that the blockage of ARF1 activation and the disruption of ARF1 localization to the Golgi by mutating Thr48, a highly conserved residue involved in the exchange of GDP for GTP, and the myristoylation site Gly2 abolished ARF1's ability to activate ERK1/2. In addition, treatment with Golgi structure disrupting agents markedly attenuated ARF1-mediated ERK1/2 activation. Furthermore, ARF1 significantly promoted cell proliferation. More interestingly, ARF1 activated 90kDa ribosomal S6 kinase 1 (RSK1) without influencing Elk-1 activation and ERK2 translocation to the nuclei. These data demonstrate that, once activated, ARF1 activates the MAPK pathway likely using the Golgi as a main platform, which in turn activates the cytoplasmic RSK1, leading to cell proliferation.

Central GLP-2 enhances hepatic insulin sensitivity via activating PI3K signaling in POMC neurons.

Glucagon-like peptides (GLP-1/GLP-2) are coproduced and highlighted as key modulators to improve glucose homeostasis and insulin sensitivity after bariatric surgery. However, it is unknown if CNS GLP-2 plays any physiological role in the control of glucose homeostasis and insulin sensitivity. We show that mice lacking GLP-2 receptor (GLP-2R) in POMC neurons display glucose intolerance and hepatic insulin resistance. GLP-2R activation in POMC neurons is required for GLP-2 to enhance insulin-mediated suppression of hepatic glucose production (HGP) and gluconeogenesis. GLP-2 directly modulates excitability of POMC neurons in GLP-2R- and PI3K-dependent manners. GLP-2 initiates GLP-2R-p85α interaction and facilitates PI3K-Akt-dependent FoxO1 nuclear exclusion in POMC neurons. Central GLP-2 suppresses basal HGP and enhances insulin sensitivity, which are abolished in POMC-p110α KO mice. Thus, CNS GLP-2 plays a key physiological role in the control of HGP through activating PI3K-dependent modulation of membrane excitability and nuclear transcription of POMC neurons in the brain.

Differential regulation of the cell-surface targeting and function of beta- and alpha1-adrenergic receptors by Rab1 GTPase in cardiac myocytes.

The molecular mechanism underlying the export from the endoplasmic reticulum (ER) to the cell surface and its role in the regulation of signaling of adrenergic receptors (ARs) remain largely unknown. In this report, we determined the role of Rab1, a Ras-like GTPase that coordinates protein transport specifically from the ER to the Golgi, in the cell surface targeting and function of endogenous beta- and alpha1-ARs in neonatal rat ventricular myocytes. Adenovirus-driven expression of Rab1 into myocytes selectively increased the cell-surface number of alpha1-AR, but not beta-AR, whereas the dominant-negative mutant Rab1N124I significantly reduced the cell-surface expression of beta-AR and alpha1-AR. Brefeldin A inhibited beta-AR and alpha1-AR export and antagonized the Rab1 effect on alpha1-AR expression. Manipulation of Rab1 function similarly influenced the transport of alpha1A- and alpha1B-ARs as well as beta1- and beta2-ARs. Fluorescent microscopy analysis demonstrated that expression of Rab1N124I and Rab1 small interfering RNA induced a marked accumulation of GFP-tagged beta2-AR and alpha1B-AR in the ER. Consistent with the effects on receptor cell-surface targeting, Rab1 selectively enhanced ERK1/2 activation and hypertrophic growth in response to the alpha1-AR agonist phenylephrine but not to the beta-AR agonist isoproterenol. Rab1N124I inhibited both agonist-mediated ERK1/2 activation and hypertrophic growth in neonatal myocytes. These results demonstrate that the cell-surface targeting and signaling of beta- and alpha1-ARs require Rab1 and are differentially modulated by augmentation of Rab1 function. Our data provide strong evidence implicating the ER-to-Golgi traffic as a site for selective manipulation of distinct AR function in cardiac myocytes.

Enhancement of the recycling and activation of beta-adrenergic receptor by Rab4 GTPase in cardiac myocytes.

We investigate the role of Rab4, a Ras-like small GTPase coordinating protein transport from the endosome to the plasma membrane, on the recycling and activation of endogenous beta-adrenergic receptor (beta-AR) in HL-1 cardiac myocytes in vitro and transgenic mouse hearts in vivo. Beta1-AR, the predominant subtype of beta-AR in HL-1 cardiac myocytes, was internalized after stimulation with isoproterenol (ISO) and fully recycled at 4 h upon ISO removal. Transient expression of Rab4 markedly facilitated recycling of internalized beta-AR to the cell surface and enhanced beta-AR signaling as measured by ISO-stimulated cAMP production. Transgenic overexpression of Rab4 in the mouse myocardium significantly increased the number of beta-AR in the plasma membrane and augmented cAMP production at the basal level and in response to ISO stimulation. Rab4 overexpression induced concentric cardiac hypertrophy with a moderate increase in ventricle/body weight ratio and posterior wall thickness and a selective up-regulation of the beta-myosin heavy chain gene. These data provide the first evidence indicating that Rab4 is a rate-limiting factor for the recycling of endogenous beta-AR and augmentation of Rab4-mediated traffic enhances beta-AR function in cardiac myocytes.

A conserved motif for the transport of G protein-coupled receptors from the endoplasmic reticulum to the cell surface.

The structural determinants for the export trafficking of G protein-coupled receptors are poorly defined. In this report, we determined the role of carboxyl termini (CTs) of alpha2B-adrenergic receptor (AR) and angiotensin II type 1A receptor (AT1R) in their transport from the endoplasmic reticulum (ER) to the cell surface. The alpha2B-AR and AT1R mutants lacking the CTs were completely unable to transport to the cell surface and were trapped in the ER. Alanine-scanning mutagenesis revealed that residues Phe436 and Ile433-Leu444 in the CT were required for alpha2B-AR export. Insertion or deletion between Phe436 and Ile443-Leu444 as well as Ile443-Leu444 mutation to FF severely disrupted alpha2B-AR transport, indicating there is a defined spatial requirement, which is essential for their function as a single motif regulating receptor transport from the ER. Furthermore, the carboxyl-terminally truncated as well as Phe436 and Ile443-Leu444 mutants were unable to bind ligand and the alpha2B-AR CT conferred its transport properties to the AT1R mutant without the CT in a Phe436-Ile443-Leu444-dependent manner. These data suggest that the Phe436 and Ile443-Leu444 may be involved in both proper folding and export from the ER of the receptor. Similarly, residues Phe309 and Leu316-Leu317 in the CT were identified as essential for AT1R export. The sequence F(X)6LL (where X can be any residue, and L is leucine or isoleucine) is highly conserved in the membrane-proximal CTs of many G protein-coupled receptors and may function as a common motif mediating receptor transport from the ER to the cell surface.

Regulation of the cell surface expression and function of angiotensin II type 1 receptor by Rab1-mediated endoplasmic reticulum-to-Golgi transport in cardiac myocytes.

Rab1 GTPase coordinates vesicle-mediated protein transport specifically from the endoplasmic reticulum (ER) to the Golgi apparatus. We recently demonstrated that Rab1 is involved in the export of angiotensin II (Ang II) type 1 receptor (AT1R) to the cell surface in HEK293 cells and that transgenic mice overexpressing Rab1 in the myocardium develop cardiac hypertrophy. To expand these studies, we determined in this report whether the modification of Rab1-mediated ER-to-Golgi transport can alter the cell surface expression and function of endogenous AT1R and AT1R-mediated hypertrophic growth in primary cultures of neonatal rat ventricular myocytes. Adenovirus-mediated gene transfer of wild-type Rab1 (Rab1WT) significantly increased cell surface expression of endogenous AT1R in neonatal cardiomyocytes, whereas the dominant-negative mutant Rab1N124I had the opposite effect. Brefeldin A treatment blocked the Rab1WT-induced increase in AT1R cell surface expression. Fluorescence analysis of the subcellular localization of AT1R revealed that Rab1 regulated AT1R transport specifically from the ER to the Golgi in HL-1 cardiomyocytes. Consistent with their effects on AT1R export, Rab1WT and Rab1N124I differentially modified the AT1R-mediated activation of ERK1/2 and its upstream kinase MEK1. More importantly, adenovirus-mediated expression of Rab1N124I markedly attenuated the Ang II-stimulated hypertrophic growth as measured by protein synthesis, cell size, and sarcomeric organization in neonatal cardiomyocytes. In contrast, Rab1WT expression augmented the Ang II-mediated hypertrophic response. These data strongly indicate that AT1R function in cardiomyocytes can be modulated through manipulating AT1R traffic from the ER to the Golgi and provide the first evidence implicating the ER-to-Golgi transport as a regulatory site for control of cardiomyocyte growth.