Expression of Rab3D N135I Inhibits Regulated Secretion of ACTH in AtT-20 Cells

Rab proteins are a family of small GTPases that regulate intracellular vesicle traffic. Rab8b, because of its homology with Rab8, has been suggested to function in vesicle transport to the plasma membrane. Using the yeast two-hybrid system, we identified a Rab8b interacting clone, termed TRIP8b, from a rat brain cDNA library. The gene encodes a 66-kDa protein with homology to the peroxisomal targeting signal 1 receptor. The interaction between Rab8b and TRIP8b was further verified by in vitro binding assays and co-immunoprecipitation studies. Additional experiments with Rab8b mutants demonstrated that Rab8b requires a guanine nucleotide but not prenylation for its interaction with TRIP8b. Western immunoblot analysis showed that TRIP8b was primarily expressed in brain. Subcellular fractionation of AtT20 cells revealed that TRIP8b was present in both cytosolic and membrane fractions. To investigate the function of Rab8b and TRIP8b in secretion, we examined the release of ACTH from AtT20 cells. Results from stable cell lines expressing Rab8b or TRIP8b indicated that both proteins had a stimulatory effect on cAMP-induced secretion of ACTH. In summary, these data suggest that Rab8b and TRIP8b interact with each other and are involved in the regulated secretory pathway in AtT20 cells.Rab proteins are a family of small GTP-binding proteins that are important regulators of vesicle transport (1-3). They undergo nucleotide exchange to establish the active GTP-bound form and are incorporated onto transport vesicles either during or after vesicle formation. The GTP-bound form of Rab proteins recruit effectors, either directly or indirectly, to target vesicles to the appropriate sites on acceptor membranes. These effectors include motor proteins which link the vesicles to the cytoskeleton (4, 5), docking complexes which are recruited from the cytosol or tethering factors that mediate the initial contact of membranes that are destined to fuse (6, 7).To date, about 40 distinct Rab proteins have been identified and each is believed to be specifically associated with a particular vesicle transport pathway (2,8). However, only a fraction of known Rab proteins have their functions characterized in detail. Among these, Rab8 (which we term Rab8a) has been shown to be a key regulator of constitutive polarized vesicle transport to the dendrites in the neurons or to the basolateral membrane in epithelial cells (9 -11). Using immunofluorescence and electron microscopy, Rab8a is localized to the Golgi region, cytoplasmic vesicular structures, and the plasma membrane of Madine-Darby canine kidney epithelial cells. When the wild type and dominant active mutant forms of Rab8a are overexpressed in baby hamster kidney fibroblast cells, a dramatic change in cell morphology occurs. The cells form elongated processes as a result of a reorganization of both their actin filaments and microtubules. In this case, newly synthesized vesicular stomatitis virus, a basolateral marker protein is preferentially delivered into these cell outgrowths.While th...

Section: Rab8b and Trip8b Stimulate Secretion Of Acth From Att20supporting

confidence: 83%

supporting

confidence: 91%

See 1 more Smart Citation

Rab8b and Its Interacting Partner TRIP8b Are Involved in Regulated Secretion in AtT20 Cells

Chen¹,

Liang²,

Chia³

et al. 2001

“…Immunoelectron Microscopy-Immunogold labeling of broken and agarose-embedded AtT-20 cells was done as described elsewhere (25). Rabbit polyclonal anti-syndet antibodies were used at 1:20 dilution and mouse monoclonal anti-SNAP-25 antibodies were used at 1:100 dilution.…”

Section: Methodsmentioning

confidence: 99%

Role of the Cysteine-rich Domain of the t-SNARE Component, SYNDET, in Membrane Binding and Subcellular Localization

Koticha

Huddleston²,

Witkin

et al. 1999

Self Cite

Wild-type syndet is efficiently recruited at the plasma membrane in transfected AtT-20 cells. A deletion at the cysteine-rich domain abolishes palmitoylation, membrane binding, and plasma membrane distribution of syndet. Syndet, SNAP-25A, and SNAP-25B share four cysteine residues, of which three, Cys 2 , Cys 4 , and Cys 5 , are absolutely conserved in all three homologs. Mutations at any pair of cysteines within cysteines 2, 4, and 5 shift syndet from the cell surface into the cytoplasm. Thus, at least two cysteines within the conserved triplet are necessary for plasma membrane localization. Syndet C1S/C3S, with substitutions at the pair Cys 1 and Cys 3 , distributes to the plasma membrane, a Golgi-like compartment, and the cytosol. We conclude that Cys 1 and Cys 3 are not absolutely necessary for membrane binding or plasma membrane localization. Our results show that the cysteine-rich domain of syndet plays a major role in its subcellular distribution.SNAP-25 homologues belong to a small family of structurally similar proteins thought to function in neurotransmission and axonal growth (1-5). SNAP-25 is very abundant in neurons where it localizes to presynaptic membranes and synaptic vesicles (1, 6). It has been shown that SNAP-25 and syntaxin-1, two proteins localized at the plasma membrane, form a complex with VAMP-2, an integral membrane protein of synaptic vesicles (3). This heterotrimeric complex is a receptor (SNARE) for SNAP and NSF, two soluble factors required for vesicular traffic (3). A current model predicts that SNARE receptors at an acceptor compartment (t-SNAREs) interact in a "lock and key" fashion with SNARE receptors at the surface of transport vesicles (v-SNAREs) (reviewed in Ref. 7). Recently, it has been proposed that SNARE components, by themselves, are sufficient to mediate fusion between two lipid bilayers (8) and pairing of specific v-SNAREs and t-SNAREs determines specificity of organelle traffic. In agreement with this hypothesis, there are multiple members of the VAMP and syntaxin family of proteins, each localized at a specific intracellular compartment (9). A homolog of SNAP-25, called SNAP-23, was identified from a human B lymphocyte library by yeast double-hybrid screening (10).

“…In Aplysia and in Lymnaea, distinct classes of large dense core vesicles containing different products of pro-ELH processing are released into the circulation from axon endings or into extracellular space from collaterals (61)(62)(63). The large dense core vesicles of atrial myocytes are localized to the perinuclear region (64,65), whereas corticotrope vesicles accumulate under the plasma membrane (39,66,67), and the vesicles in hypothalamic neurosecretory neurons are transported long distances to terminals in the neural lobe of the pituitary. and Kalirin were rinsed with complete serum-free medium and incubated with complete serum-free medium for three sequential 30-min periods before the experiment was started.…”

Section: Expression Of Kalirin Alters Pam/antibody Internalization Bymentioning

confidence: 99%

Kalirin, a Multifunctional PAM COOH-terminal Domain Interactor Protein, Affects Cytoskeletal Organization and ACTH Secretion from AtT-20 Cells

Mains

Alam

Johnson

et al. 1999

The production and regulated secretion of bioactive peptides require a series of lumenal enzymes to convert inactive precursors into bioactive peptides plus several cytosolic proteins to govern granule formation, maturation, translocation, and exocytosis. Peptidylglycine ␣-amidating monooxygenase (PAM), an enzyme essential for biosynthesis of many peptides, is an integral membrane protein with trafficking information in both its lumenal and cytosolic domains. Kalirin, a PAM cytosolic domain interactor protein with spectrin-like repeats and GDP/GTP exchange factor activity for Rac1, is