Patricia K. Curran

The Cl - /H + antiporter ClC-7 is the primary chloride permeation pathway in lysosomes

Lysosomes are the stomachs of the cell—terminal organelles on the endocytic pathway where internalized macromolecules are degraded. Containing a wide range of hydrolytic enzymes, lysosomes depend on maintaining acidic luminal pH values for efficient function. Although acidification is mediated by a V-type proton ATPase, a parallel anion pathway is essential to allow bulk proton transport. The molecular identity of this anion transporter remains unknown. Recent results of knockout experiments raise the possibility that ClC-7, a member of the CLC family of anion channels and transporters, is a contributor to this pathway in an osteoclast lysosome-like compartment, with loss of ClC-7 function causing osteopetrosis. Several mammalian members of the CLC family have been characterized in detail; some (including ClC-0, ClC-1 and ClC-2) function as Cl--conducting ion channels, whereas others act as Cl-/H+antiporters (ClC-4 and ClC-5). However, previous attempts at heterologous expression of ClC-7 have failed to yield evidence of functional protein, so it is unclear whether ClC-7 has an important function in lysosomal biology, and also whether this protein functions as a Cl- channel, a Cl-/H+ antiporter, or as something else entirely. Here we directly demonstrate an anion transport pathway in lysosomes that has the defining characteristics of a CLC Cl-/H+ antiporter and show that this transporter is the predominant route for Cl- through the lysosomal membrane. Furthermore, knockdown of ClC-7 expression by short interfering RNA can essentially ablate this lysosomal Cl-/H+ antiport activity and can strongly diminish the ability of lysosomes to acidify in vivo, demonstrating that ClC-7 is a Cl-/H+ antiporter, that it constitutes the major Cl- permeability of lysosomes, and that it is important in lysosomal acidification.

Differences in endosomal targeting of human β1- and β2-adrenergic receptors following clathrin-mediated endocytosis

The β2-adrenergic receptor (β2AR) undergoes agonist-mediated endocytosis via clathrin-coated pits by a process dependent on both arrestins and dynamin. Internalization of some G protein-coupled receptors, however, is independent of arrestins and/or dynamin and through other membrane microdomains such as caveolae or lipid rafts. The human β1AR is less susceptible to agonist-mediated internalization than the β2-subtype, and its endocytic route, which is unknown, may be different. We have found that (i) co-expression of arrestin-2 or -3 enhanced the internalization of both subtypes whereas co-expression of dominant-negative mutants of arrestin-2 or dynamin impaired their internalization, as did inhibitors of clathrin-mediated endocytosis. (ii) Agonist stimulation increased the phosphorylation of β2AR but not β1AR. (iii) In response to agonist, each subtype redistributed from the cell surface to a distinct population of cytoplasmic vesicles; those containing β1AR were smaller and closer to the plasma membrane whereas those containing β2AR were larger and more perinuclear. (iv) When subcellular fractions from agonist-treated cells were separated by sucrose density gradient centrifugation, all of the internalized β2AR appeared in the lighter endosomal-containing fractions whereas some of the internalized β1AR remained in the denser plasma membrane-containing fractions. (v) Both subtypes recycled with similar kinetics back to the cell surface upon removal of agonist; however, recycling of β2AR but not β1AR was inhibited by monensin. Based on these results, we propose that the internalization of β1AR is both arrestin- and dynamin-dependent and follows the same clathrin-mediated endocytic pathway as β2AR. But during or after endocytosis, β1AR and β2AR are sorted into different endosomal compartments.

Endogenous β3- but not β1-adrenergic receptors are resistant to agonist-mediated regulation in human SK-N-MC neurotumor cells

Abstract Although there is considerable interest in the regulation of the different β-adrenergic receptor (AR) subtypes, most previous studies have utilized stably transfected cells expressing recombinant receptors under the control of viral promoters. Human SK-N-MC neurotumor cells appear to be novel, since they express both endogenous β 1 AR and β 3 AR based on radioligand binding and on functional response. Saturation binding of either the hydrophilic ligand (−)-[ 3 H]CGP-12177 or the more hydrophobic (−)-[ 125 I]iodocyanopindolol indicated the presence of two populations of binding sites with high and low affinities. With either ligand, the β 1 AR antagonist CGP-20712A preferentially inhibited binding to the high-affinity sites. This is consistent with the latter representing β 1 AR whereas the low-affinity sittes represent β 3 AR. Both subtypes appeared to be functional on the basis of isoproterenol stimulation of cyclic adenosine monophosphate (cAMP) in intact cells and adenylyl cyclase activity in cell membranes in the absence and presence of CGP-20712A. SK-N-MC-IXC cells, derived by twice subcloning the parental cells, also expressed both βAR subtypes, indicating that they co-exist in the same cell. SK-N-MC cells exposed to isoproterenol exhibited a rapid sequestration and a slower downregulation of β 1 AR. The latter subtype also underwent desensitization, as indicated by a rightward shift to less sensitivity in the EC 50 for isoproterenol stimulation of adenylyl cyclase activity. In contrast, the β 3 AR subtype was resistant to agonist-mediated sequestration, downregulation, and desensitization. Thus, when endogenously expressed in the same cell line, human β 1 AR and β 3 AR display differences in their ability to be regulated by agonist.

Brefeldin A inhibits protein synthesis in cultured cells

The fungal metabolite brefeldin A (BFA) is known to disrupt the Golgi apparatus resulting in redistribution of Golgi proteins to the endoplasmic reticulum and inhibition of protein secretion. BFA was found to inhibit protein synthesis in rat glioma C6 cells by up to 70% between 0.1 and 1,μg/ml. Inhibition was both time‐dependent and reversible. BFA inhibited protein synthesis to varying degrees in a number of other cell lines but not in BFA‐resistant marsupial kidney cells. The same concentrations of BFA which inhibited protein synthesis, also blocked the inhibitory effects of Pseudomonas exotoxin and ricin on BFA‐sensitive cells. BFA, however, was unable to block the inhibition of protein synthesis by the toxins in the resistant marsupial kidney cells.

Modulation of Adenylylcyclase by Protein Kinase C in Human Neurotumor SK‐N‐MC Cells: Evidence that the α Isozyme Mediates Both Potentiation and Desensitization

Abstract: Exposure of human SK‐N‐MC neurotumor cells to 4β‐phorbol 12‐myristate 13‐acetate (PMA) increased isoproterenol stimulation of cyclic AMP levels by severalfold. This potentiation was blocked by inhibitors of protein kinase C (PKC) and did not occur in cells in which PKC had been down‐regulated. PMA treatment also enhanced the stimulation by dopamine, cholera toxin, and forskolin. Thus, the effect of PMA on the adenylylcyclase system was postreceptor and involved either the guanine nucleotide binding regulatory (G) proteins or the cyclase itself. As PMA treatment did not impair the inhibition of isoproterenol stimulation by neuropeptide Y, an involvement of the inhibitory G protein Gi was unlikely. Cholate extracts of membranes from control and PMA‐treated cells were equally effective in the reconstitution of adenylylcyclase activity in S49 cyc− membranes, which lack the stimulatory G protein subunit Gsα; thus, Gs did not appear to be the target of PMA action. Membranes from PMA‐treated cells exhibited increased adenylylcyclase activity to all stimulators including Mn2+ and Mn2+ plus forskolin. In addition, activity was increased when control membranes were incubated with ATP and purified PKC from rat brain. This is consistent with a direct effect of PKC on the adenylylcyclase catalyst in SK‐N‐MC cells. PMA treatment also resulted in a shift to less sensitivity in the Kact for isoproterenol but not for dopamine or CGP‐12177 (a β3‐adrenergic agonist) stimulation. Thus, the β1 but not the D1 or β3 receptors were being desensitized by PKC activation. Analysis of SK‐N‐MC cells by western blotting with antibodies against different PKC isozymes revealed that both the α and ζ isozymes were present in these cells. Whereas PKC‐α was activated and translocated from cytosol to membrane by phorbol esters, the ζ isozyme was not. Thus, PKC‐α, which has been implicated in desensitization in other cell lines, also appears to potentiate adenylylcyclase activity.

Independent and Coordinate Regulation of β1- and β2-Adrenergic Receptors in Rat C6 Glioma Cells

Rat C6 glioma cells have both beta 1- and beta 2-adrenergic receptors in approximately 7:3 ratio. When the cells were exposed to the beta-adrenergic agonist isoproterenol, there was a rapid sequestration of up to 50% of the surface receptor population over a 30-min period as measured by the loss of binding of the hydrophilic ligand [3H] CGP-12177 to intact cells. Using the beta 1-selective antagonist CGP 20712A to quantify the proportion of the two subtypes, it was found that although both beta 1 and beta 2 receptors were sequestered, the latter were sequestered initially twice as fast as the former. More prolonged agonist exposure led to a down-regulation of approximately 90% of the total receptor population by 6 h as measured by the loss of binding of the more hydrophobic ligand [125I]iodocyanopindolol to cell lysates. The two subtypes, however, underwent down-regulation with similar kinetics. Treatment of the cells with agents that raise cyclic AMP levels such as cholera toxin and forskolin resulted in a slower, but still coordinated down-regulation of both subtypes. Thus, there appears to be both independent and coordinate regulation of endogenous beta 1-and beta 2-adrenergic receptors in the same cell line.