James E. Casanova

ARNO Is a Guanine Nucleotide Exchange Factor for ADP-ribosylation Factor 6

ADP-ribosylation factors (ARFs) constitute a family of small monomeric GTPases. ARFs 1 and 3 function in the recruitment of coat proteins to membranes of the Golgi apparatus, whereas ARF6 is localized to the plasma membrane, where it appears to modulate both the assembly of the actin cytoskeleton and endocytosis. Like other GTPases, ARF activation is facilitated by specific guanine nucleotide exchange factors (GEFs). ARNO (ARF nucleotide-binding site opener) is a member of a growing family of ARF-GEFs that share a common, tripartite structure consisting of an N-terminal coiled-coil domain, a central domain with homology to the yeast protein Sec7p, and a C-terminal pleckstrin homology domain. Recently, ARNO and its close homologue cytohesin-1 were found to catalyze in vitronucleotide exchange on ARF1 and ARF3, respectively, raising the possibility that these GEFs function in the Golgi. However, the actual function of these proteins may be determined in part by their ability to interact with specific ARFs and in part by their subcellular localization. We report here that in vitro ARNO can stimulate nucleotide exchange on both ARF1 and ARF6. Furthermore, based on subcellular fractionation and immunolocalization experiments, we find that ARNO is localized to the plasma membrane in mammalian cells rather than the Golgi. It is therefore likely that ARNO functions in plasma membrane events by modulating the activity of ARF6 in vivo. These findings are consistent with the previous observation that cytohesin-1 regulates the adhesiveness of αLβ2integrins at the plasma membrane of lymphocytes.

Intra-endosomal pH-sensitive Recruitment of the Arf-nucleotide Exchange Factor ARNO and Arf6 from Cytoplasm to Proximal Tubule Endosomes

Kidney proximal tubule epithelial cells have an extensive apical endocytotic apparatus that is critical for the reabsorption and degradation of proteins that traverse the glomerular filtration barrier and that is also involved in the extensive recycling of functionally important apical plasma membrane transporters. We show here that an Arf-nucleotide exchange factor, ARNO (ADP-ribosylation factor nucleotide site opener) as well as Arf6 and Arf1 small GTPases are located in the kidney proximal tubule receptor-mediated endocytosis pathway, and that ARNO and Arf6 recruitment from cytosol to endosomes is pH-dependent. In proximal tubules in situ, ARNO and Arf6 partially co-localized with the V-ATPase in apical endosomes in proximal tubules. Arf1 was localized both at the apical pole of proximal tubule epithelial cells, but also in the Golgi. By Western blot analysis ARNO, Arf6, and Arf1 were detected both in purified endosomes and in proximal tubule cytosol. A translocation assay showed that ATP-driven endosomal acidification triggered the recruitment of ARNO and Arf6 from proximal tubule cytosol to endosomal membranes. The translocation of both ARNO and Arf6 was reversed by V-type ATPase inhibitors and by uncouplers of endosomal intralumenal pH, and was correlated with the magnitude of intra-endosomal acidification. Our data suggest that V-type ATPase-dependent acidification stimulates the selective recruitment of ARNO and Arf6 to proximal tubule early endosomes. This mechanism may play an important role in the pH-dependent regulation of receptor-mediated endocytosis in proximal tubulesin situ.

Identification of a Plasma Membrane-associated Guanine Nucleotide Exchange Factor for ARF6 in Chromaffin Cells POSSIBLE ROLE IN THE REGULATED EXOCYTOTIC PATHWAY

ADP-ribosylation factors (ARFs) constitute a family of structurally related proteins that forms a subset of the Ras superfamily of regulatory GTP-binding proteins. Like other GTPases, activation of ARFs is facilitated by specific guanine nucleotide exchange factors (GEFs). In chromaffin cells, ARF6 is associated with the membrane of secretory granules. Stimulation of intact cells or direct elevation of cytosolic calcium in permeabilized cells triggers the rapid translocation of ARF6 to the plasma membrane and the concomitant activation of phospholipase D (PLD) in the plasma membrane. Both calcium-evoked PLD activation and catecholamine secretion in permeabilized cells are strongly inhibited by a synthetic peptide corresponding to the N-terminal domain of ARF6, suggesting that the ARF6-dependent PLD activation near the exocytotic sites represents a key event in the exocytotic reaction in chromaffin cells. In the present study, we demonstrate the occurrence of a brefeldin A-insensitive ARF6-GEF activity in the plasma membrane and in the cytosol of chromaffin cells. Furthermore, reverse transcriptase-polymerase chain reaction and immunoreplica analysis indicate that ARNO, a member of the brefeldin A-insensitive ARF-GEF family, is expressed and predominantly localized in the cytosol and in the plasma membrane of chromaffin cells. Using permeabilized chromaffin cells, we found that the introduction of anti-ARNO antibodies into the cytosol inhibits, in a dose-dependent manner, both PLD activation and catecholamine secretion in calcium-stimulated cells. Furthermore, co-expression in PC12 cells of a catalytically inactive ARNO mutant with human growth hormone as a marker of secretory granules in transfected cells resulted in a 50% inhibition of growth hormone secretion evoked by depolarization with high K+. The possibility that the plasma membrane-associated ARNO participates in the exocytotic pathway by activating ARF6 and downstream PLD is discussed.

Tubulin-Associated Calmodulin-Dependent Kinase: Evidence for an Endogenous Complex of Tubulin with a Calcium-Calmodulin-Dependent Kinase

A Ca2+‐calmodulin kinase that phosphorylates tubulin and microtubule‐associated proteins as major substrates has been purified and characterized from brain cytoplasm. It is important to determine if cytoskeletal proteins are major natural substrates for this kinase system. This report demonstrates that a significant fraction of brain cytosolic calmodulin‐dependent kinase activity exists in tight association with tubulin in the form of a stable complex. The tubulin‐calmodulin kinase complex displayed an apparent molecular weight on gel filtration of approximately 1.8 × 106 daltons. The specific activity of tubulin kinase in the complex was enriched over 20‐fold in comparison with brain cytosol. Although purified tubulin alone did not adhere to a calmodulin column, the tubulin associated with the calmodulin kinase complex did bind specifically to the calmodulin affinity resin. The kinase activity was shown to be tightly associated in complex with tubulin by (1) copurification, (2) isolation on gel filtration chromatography, (3) isolation on ion‐exchange chromatography, and (4) binding to calmodulin. The kinase complexed with tubulin was identical to the previously purified kinase as judged by several criteria including (1) subunit molecular weights, (2) isoelectric points, (3) autophosphorylation characteristics, (4) calmodulin binding properties, (5) kinetic parameters of tubulin phosphorylation, (6) phosphoamino acid phosphorylation sites on α‐and β‐tubulin, and (7) identical subunit 125I‐tryptic peptide maps. The results indicate that a significant fraction of this previously purified calmodulin kinase is endogenously associated with tubulin in brain cytoplasm and may play a role in mediating some of the effects of calcium on neuronal function.

23 - Expression and Properties of Rab25 in Polarized Madin-Darby Canine Kidney Cells

This chapter discusses the expression and properties of Rab25 in polarized Madin-Darby canine kidney cells. In Madin-Darby canine kidney (MDCK) cells, Rab25 levels are very low, whereas endogenous Rablla levels are relatively high. Therefore, to study Rab25 in MDCK cells, cell lines are isolated stably transfected with the wild-type sequence of rabbit Rab25. To detect Rab25, monoclonal antibodies are developed specific for Rab25 and nonreactive against Rab11a. A double-screening enzyme-linked immunosorbent assay (ELISA) protocolis developed to isolate monoclonal antibodies against Rab25 that do not react with Rablla. To visualize the three-dimensional distribution of Rab proteins in polarized cells, the stably transfected Rab25- expressing 2A3 cell line is grown to confluence on Transwell clear filters and maintained for three days following confluence. Out of 20 lines screened, six lines were found to contain a transfected Rab25 message. The 2A3 line utilized for characterization of the association of Rablla and Rab259 was one of the two lowest expressing cell lines. To compare the effects of stable overexpression of Rab25 on endogenous message levels, Rab25 and Rablla mRNA levels were compared between mock pCB6-transfected cells and the Rab25-transfected 2A3 line.

Expression and analysis of ARNO and ARNO mutants and their effects on ADP-ribosylation factor (ARF)-mediated actin cytoskeletal rearrangements.

This chapter discusses expression and analysis of ARNO and ARNO mutants and their effects on adenosine diphosphate (ADP)-ribosylation factor (ARF)-mediated actin cytoskeletal rearrangements. To facilitate detection of ARNO mutants in the presence of the endogenous protein, an N-terminal myc epitope tag (EQKLISEEDL) is introduced by polymerase chain reaction (PCR) mutagenesis. Constructs tagged at the C terminus behave identically to those with N terminal tags. These tagged forms of ARF have been extensively characterized and behave identically to the native proteins in all aspects tested to date. Unlike Ras or Rho family GTPases, the intrinsic (non-GAP-mediated) rate of GTP hydrolysis among ARF family members is quite low. It is therefore possible to quantitate the GTP-GDP ratio of individual ARFs by extraction of bound nucleotide from immunoprecipitates from metabolically labeled cells. For morphologic analysis of cells expressing ARNO and ARNO mutants, HeLa cells are cultured on glass coverslips, and transfected 24–48 hr prior to analysis, depending on the level of expression desired. ARNO contains a consensus site for phosphorylation by protein kinase C (PKC) at its C terminus. To determine the effects of PKC activation on ARNO function, a subset of cultures is treated with phorbol 13-myristate acetate (PMA) for 30 min prior to fixation. PMA is maintained as a 1 mM stock in DMSO at –20°C.

Structure and Function of the Polymeric Immunoglobulin Receptor in Epithelial Cells

Publisher Summary A great deal of progress has been made in the last 15 years in the understanding of the structure and function of the pIgR. The complex cellular itinerary of the pIgR was first elucidated in rat liver, and has since been found to be similar in other tissues and cultured cells. The primary structure of the pIgR has been determined by cDNA cloning and/or direct protein sequencing from rabbit, rat, human, bovine, and murine sources. The receptors from all five species are approximately 65% homologous, although higher degrees of homology are observed in regions with conserved function. Newly synthesized receptors are transported to the basolateral cell surface where ligand is bound. It should be noted that all subsequent transport processes occur independently of receptor occupancy. Receptor/ligand complexes are then rapidly internalized in clathrin-coated pits and enter an acidic endosomal compartment, along with other receptors and their respective ligands. In the relatively low pH of this compartment many ligands dissociate from their receptors; however, IgA remains tightly bound under these conditions. Ultrastructural studies have revealed that the pIgR becomes sequestered in a subcompartment of the endosome, where it is packaged into transcytotic carrier vesicles. Transport of these vesicles to the apical cell surface appears to be facilitated by microtubules, as treatment of cells with nocodazole or other microtubule-depolymerizing agents substantially inhibits transport. At some point during the transport process, a disulfide bond forms between the receptor and one of the IgA monomers, linking them covalently. Upon arrival at the apical plasma membrane, the extracellular domain of the receptor is proteolytically cleaved, producing SC which, along with bound IgA, is released into secretions.

Transepithelial Transport of Polymeric Immunoglobulins

Epithelial cells serve as a selectively permeable barrier between the lumenal and serosal compartments of a wide variety of tissues. While the movement of small molecules such as water, ions and sugars across epithelia has been the object of intense study for many years, it is becoming increasingly apparent that macromolecules ( i . e . , proteins) can also be subjected to active transepithelial transport. A growing number of proteins have been demonstrated to undergo transcytosis, including EGF,’ NGF,’ prolactin3 and beta-lact~globulin.~ Perhaps the best known example of this process is the transport of immunoglobulins, which occurs in several situations in mammals: 1) transport of maternal IgG across the fetal yolk sac or the intestinal epithelium in neonatal rats, and across the placenta in humans; 2) transport of the polymeric immunoglobulins IgA and IgM (pIg) across a variety of mucosal tissues. Both are receptor-mediated processes that take place in opposite directions across the epithelial layer; IgG is transported in the apical to basolateral direction, IgA and IgM from basolateral to apical. Of these, the most thoroughly understood is the transport of secretory IgA. In this paper, we shall discuss what is currently known about the cellular and molecular basis of IgA transport, and how this phenomenon relates to the general process of membrane traffic in epithelia.