Harish Radhakrishna

Transfer of M2 Muscarinic Acetylcholine Receptors to Clathrin-derived Early Endosomes following Clathrin-independent Endocytosis

Upon agonist stimulation, many G protein-coupled receptors such as β2-adrenergic receptors are internalized via β-arrestin- and clathrin-dependent mechanisms, whereas others, like M2 muscarinic acetylcholine receptors (mAChRs), are internalized by clathrin- and arrestin-independent mechanisms. To gain further insight into the mechanisms that regulate M2 mAChR endocytosis, we investigated the post-endocytic trafficking of M2 mAChRs in HeLa cells and the role of the ADP-ribosylation factor 6 (Arf6) GTPase in regulating M2 mAChR internalization. Here, we report that M2 mAChRs are rapidly internalized by a clathrin-independent pathway that is inhibited up to 50% by expression of either GTPase-defective Arf6 Q67L or an upstream Arf6 activator, Gαq Q209L. In contrast, M2mAChR internalization was not affected by expression of dominant-negative dynamin 2 K44A, which is a known inhibitor of clathrin-dependent endocytosis. Nevertheless, M2 mAChRs, which are initially internalized in structures that lack clathrin-dependent endosomal markers, quickly localize to endosomes that contain the clathrin-dependent, early endosomal markers early endosome autoantigen-1, transferrin receptor, and GTPase-defective Rab5 Q79L, which is known to swell early endosomal compartments. These results suggest that M2mAChRs initially internalize via an Arf6-associated, clathrin-independent pathway but then quickly merge with the clathrin endocytic pathway at the level of early endosomes.

Contractility modulates cell adhesion strengthening through focal adhesion kinase and assembly of vinculin-containing focal adhesions.

Actin–myosin contractility modulates focal adhesion assembly, stress fiber formation, and cell migration. We analyzed the contributions of contractility to fibroblast adhesion strengthening using a hydrodynamic adhesion assay and micropatterned substrates to control cell shape and adhesive area. Serum addition resulted in adhesion strengthening to levels 30–40% higher than serum‐free cultures. Inhibition of myosin light chain kinase or Rho‐kinase blocked phosphorylation of myosin light chain to similar extents and eliminated the serum‐induced enhancements in strengthening. Blebbistatin‐induced inhibition of myosin II reduced serum‐induced adhesion strength to similar levels as those obtained by blocking myosin light chain phosphorylation. Reductions in adhesion strengthening by inhibitors of contractility correlated with loss of vinculin and talin from focal adhesions without changes in integrin binding. In vinculin‐null cells, inhibition of contractility did not alter adhesive force, whereas controls displayed a 20% reduction in adhesion strength, indicating that the effects of contractility on adhesive force are vinculin‐dependent. Furthermore, in cells expressing FAK, inhibitors of contractility reduced serum‐induced adhesion strengthening as well as eliminated focal adhesion assembly. In contrast, in the absence of FAK, these inhibitors did not alter adhesion strength or focal adhesion assembly. These results indicate that contractility modulates adhesion strengthening via FAK‐dependent, vinculin‐containing focal adhesion assembly. J. Cell. Physiol. 223:746–756, 2010.

Separation of Membrane Trafficking and Actin Remodeling Functions of ARF6 with an Effector Domain Mutant

ABSTRACT The ADP-ribosylation factor 6 (ARF6) GTPase has a dual function in cells, regulating membrane traffic and organizing cortical actin. ARF6 activation is required for recycling of the endosomal membrane back to the plasma membrane (PM) and also for ruffling at the PM induced by Rac. Additionally, ARF6 at the PM induces the formation of actin-containing protrusions. To identify sequences in ARF6 that are necessary for these distinct functions, we examined the behavior of a chimeric protein of ARF1 and ARF6. The 1-6 chimera (with the amino half of ARF1 and the carboxyl half of ARF6) localized like ARF6 in HeLa cells and moved between the endosome and PM, but it did not form protrusions, an ARF6 effector function. Two residues in the amino-terminal half of ARF6, Q37 and S38, when substituted into the 1-6 chimera allowed protrusion formation, whereas removal of these residues from ARF6 resulted in an inability to form protrusions. Interestingly, expression of 1-6 in cells selectively inhibited protrusions induced by wild-type ARF6 but had no effect on ARF6-regulated membrane movement or Rac-induced ruffling. Thus, we have uncoupled two functions of ARF6, one involved in membrane trafficking, which is necessary for Rac ruffling, and another involved in protrusion formation.

Agonist-induced endocytosis of lysophosphatidic acid-coupled LPA1/EDG-2 receptors via a dynamin2-and Rab5-dependent pathway

Lysophosphatidic acid (LPA) is a serum-borne phospholipid that exerts a pleiotropic range of effects on cells through activation of three closely related G-protein-coupled receptors termed LPA1/EDG-2, LPA2/EDG-4 and LPA3/EDG-7. Of these receptors, the LPA1 receptor is the most widely expressed. In this study, we investigated the agonist-induced endocytosis of the human LPA1 receptor, bearing an N-terminal FLAG epitope tag, in stably transfected HeLa cells. Treatment with LPA induced the rapid endocytosis of approximately 40% of surface LPA1 within 15 minutes. Internalization was both dose dependent and LPA specific since neither lysophophatidylcholine nor sphingosine-1-phosphate induced LPA1 endocytosis. Removal of agonist following 30 minutes incubation resulted in recycling of LPA1 back to the cell surface. LPA1 internalization was strongly inhibited by dominant-inhibitory mutants of both dynamin2 (K44A) and Rab5a (S34N). In addition, both dynamin2 K44A and Rab5 S34N mildly inhibited LPA1-dependent activation of serum response factor. Finally, our results also indicate that LPA1 exhibits basal, LPA-dependent internalization in the presence of serum-containing medium.

Changes in cell morphology due to plasma membrane wounding by acoustic cavitation.

Acoustic cavitation-mediated wounding (i.e., sonoporation) has great potential to improve medical and laboratory applications requiring intracellular uptake of exogenous molecules; however, the field lacks detailed understanding of cavitation-induced morphologic changes in cells and their relative importance. Here, we present an in-depth study of the effects of acoustic cavitation on cells using electron and confocal microscopy coupled with quantitative flow cytometry. High resolution images of treated cells show that morphologically different types of blebs can occur after wounding conditions caused by ultrasound exposure as well as by mechanical shear and strong laser ablation. In addition, these treatments caused wound-induced nonlytic necrotic death resulting in cell bodies we call wound-derived perikarya (WD-P). However, only cells exposed to acoustic cavitation experienced ejection of intact nuclei and nearly instant lytic necrosis. Quantitative analysis by flow cytometry indicates that wound-derived perikarya are the dominant morphology of nonviable cells, except at the strongest wounding conditions, where nuclear ejection accounts for a significant portion of cell death after ultrasound exposure.

Lysophosphatidic Acid Decreases the Nuclear Localization and Cellular Abundance of the p53 Tumor Suppressor in A549 Lung Carcinoma Cells

Lysophosphatidic acid (LPA) is a bioactive lipid that promotes cancer cell proliferation and motility through activation of cell surface G protein–coupled receptors. Here, we provide the first evidence that LPA reduces the cellular abundance of the tumor suppressor p53 in A549 lung carcinoma cells, which express endogenous LPA receptors. The LPA effect depends on increased proteasomal degradation of p53 and it results in a corresponding decrease in p53-mediated transcription. Inhibition of phosphatidylinositol 3-kinase protected cells from the LPA-induced reduction of p53, which implicates this signaling pathway in the mechanism of LPA-induced loss of p53. LPA partially protected A549 cells from actinomycin D induction of both apoptosis and increased p53 abundance. Expression of LPA1, LPA2, and LPA3 receptors in HepG2 hepatoma cells, which normally do not respond to LPA, also decreased p53 expression and p53-dependent transcription. In contrast, neither inactive LPA1 (R124A) nor another Gi-coupled receptor, the M2 muscarinic acetylcholine receptor, reduced p53-dependent transcription in HepG2 cells. These results identify p53 as a target of LPA action and provide a new dimension for understanding how LPA stimulates cancer cell division, protects against apoptosis, and thereby promotes tumor progression. (Mol Cancer Res 2007;5(11):1201–11)

A requirement for membrane cholesterol in the β-arrestin- and clathrin-dependent endocytosis of LPA1 lysophosphatidic acid receptors

Lysophosphatidic acid (LPA) stimulates heterotrimeric G protein signaling by activating three closely related receptors, termed LPA1, LPA2 and LPA3. Here we show that in addition to promoting LPA1 signaling, membrane cholesterol is essential for the association of LPA1 with β-arrestin, which leads to signal attenuation and clathrin-dependent endocytosis of LPA1. Reduction of clathrin heavy chain expression, using small interfering RNAs, inhibited LPA1 endocytosis. LPA1 endocytosis was also inhibited in β-arrestin 1 and 2-null mouse embryo fibroblasts (β-arrestin 1/2 KO MEFs), but was restored upon re-expression of wild-type β-arrestin 2. β-arrestin attenuates LPA signaling as LPA1-dependent phosphoinositide hydrolysis was significantly elevated in β-arrestin 1/2 KO MEFs and was reduced to wild-type levels upon re-expression of wild-type β-arrestin. Interestingly, extraction of membrane cholesterol with methyl-β-cyclodextrin inhibited LPA1 signaling, β-arrestin membrane recruitment and LPA1 endocytosis. Cholesterol repletion restored all of these functions. However, neither the stimulation of phosphoinositide hydrolysis by the M1 acetylcholine receptor nor its endocytosis was affected by cholesterol extraction. LPA treatment increased the detergent resistance of LPA1 and this was inhibited by cholesterol extraction, suggesting that LPA1 localizes to detergent-resistant membranes upon ligand stimulation. These data indicate that although LPA1 is internalized by clathrin- and β-arrestin dependent endocytosis, membrane cholesterol is critical for LPA1 signaling, membrane recruitment of β-arrestins and LPA1 endocytosis.

Different Mechanisms Regulate Lysophosphatidic Acid (LPA)-dependent Versus Phorbol Ester-dependent Internalization of the LPA1 Receptor

Lysophosphatidic acid (LPA) stimulates cells by activation of five G-protein-coupled receptors, termed LPA1–5. The LPA1 receptor is the most widely expressed and is a major regulator of cell migration. In this study, we show that phorbol ester (PMA)-induced internalization of the LPA1 receptor requires clathrin AP-2 complexes, protein kinase C, and a distal dileucine motif (amino acids 352 and 353) in the cytoplasmic tail but not β-arrestin. Agonist-dependent internalization of LPA1, however, requires a cluster of serine residues (amino acids 341–347) located proximal to the dileucine motif, β-arrestin, and to a lesser extent clathrin AP-2. The serine cluster of LPA1 is required for β-arrestin2-GFP translocation to the plasma membrane and signal desensitization. In contrast, the dileucine motif (IL) is required for both basal and PMA-induced internalization. Evidence for the β-arrestin independence of PMA-induced internalization of LPA1 comes from the observations that β-arrestin2-GFP is not recruited to the plasma membrane upon PMA treatment and that LPA1 is readily internalized in β-arrestin1/2 knock-out mouse embryonic fibroblasts. These results indicate that distinct molecular mechanisms regulate agonist-dependent and PMA-dependent internalization of the LPA1 receptor.

Metabolic Labeling and Immunoprecipitation of Drosophila Proteins

The genetics of Drosophila is a powerful tool in the analysis of mutants and mutant proteins. Cultures of cells derived from wild‐type or mutant flies can be pulse labeled to biosynthetically label the proteins made by the cells. Immunoprecipitation (and subcellular fractionation) are used to characterize the expression of specific proteins.