Purification and Identification of a 28-kDa Calcium-regulated Heat-stable Protein

Self Cite

, indicating these molecules likely interact under physiological conditions. Immunofluorescence microscopy confirmed a dual localization of CRHSP-28 and annexin VI, which appeared in a punctate pattern in the supranuclear and apical cytoplasm of acini. Stimulation of cells for 5 min with the secretagogue cholecystokinin enhanced the colocalization of CRHSP-28 and annexin VI within regions of acini immediately below the apical plasma membrane. Tissue fractionation revealed that CRHSP-28 is a peripheral membrane protein that is highly enriched in smooth microsomal fractions of pancreas. Further, the content of CRHSP-28 in microsomes was significantly reduced in pancreatic tissue obtained from rats that had been infused with a secretory dose of cholecystokinin for 40 min, demonstrating that secretagogue stimulation transiently alters the association of CRHSP-28 with membranes in cells. Collectively, the Ca 2؉ -dependent binding of CRHSP-28 and annexin VI, together with their colocalization in the apical cytoplasm, is consistent with a role for these molecules in acinar cell membrane trafficking events that are essential for digestive enzyme secretion.

Section: Figmentioning

confidence: 56%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Identification of Annexin VI as a Ca2+-sensitive CRHSP-28-binding Protein in Pancreatic Acinar Cells

Thomas

Kaspar

Taft

et al. 2002

Self Cite

“…suppressant that functions by binding calcineurin, inhibits Ca 2ϩ /CaM-dependent secretion from pancreatic acinar cells, thereby implicating calmodulin/calcineurin in regulation of this secretion (42). More generally, [Ca 2ϩ ] i is clearly important in many intracellular membrane traffic events, including both regulated exocytosis of granules (43) and synaptic vesicles (44), as well as classically "constitutive" processes, such as endoplasmic reticulum to Golgi transport (45) and nuclear envelope fusion (46) (at least in in vitro systems).…”

Section: Figmentioning

confidence: 99%

Calmodulin Binds to the Basolateral Targeting Signal of the Polymeric Immunoglobulin Receptor

Chapin

Enrich

Aroeti

et al. 1996

We have identified a major calmodulin (CaM)-binding protein in rat liver endosomes using 125 I-CaM overlays from two-dimensional protein blots. Immunostaining of blots demonstrates that this protein is the polymeric immunoglobulin receptor (pIgR). We further investigated the interaction between pIgR and CaM using Madin-Darby canine kidney cells stably expressing cloned wild-type and mutant pIgR. We found that detergent-solubilized pIgR binds to CaM-agarose in a Ca 2؉ -dependent fashion, and binding is inhibited by the addition of excess free CaM or the CaM antagonist W-13 (N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide), suggesting that pIgR binding to CaM is specific. The plasma membrane of polarized epithelial cells is divided into apical and basolateral surfaces, each having a distinct protein and lipid composition. Cells use two pathways to deliver proteins to the correct surface. First, newly made proteins are packaged in the trans-Golgi network (TGN) 1 into vesicles that deliver them directly to either the apical or basolateral surface. Second, proteins can be delivered first to one surface and then endocytosed and transcytosed to the opposite surface. At least in the well polarized Madin-Darby canine kidney (MDCK) cell line, targeting to the basolateral surface generally requires a sorting signal located in the cytoplasmic domain of the protein. For a number of basolateral proteins it has been found that mutations in the cytoplasmic domain prevent TGN to basolateral targeting (1). The first basolateral signal to be identified is the 17-amino acid membrane proximal segment (residues 653-670) of the cytoplasmic domain of the polymeric immunoglobulin receptor (pIgR) (2). This protein is normally delivered from the TGN to the basolateral surface, and then endocytosed and transcytosed to the apical surface. Deletion of most of this 17-residue segment prevents TGN to basolateral delivery. Moreover, this segment can be transplanted to a heterologous reporter molecule, which is then retargeted from the apical to the basolateral surface (2). In at least one other case, the low density lipoprotein receptor, autonomous basolateral sorting signals have been identified (3).The pIgR basolateral targeting signal has been systematically analyzed by alanine scanning mutagenesis (4). Mutation of His-656, Arg-657, or Val-660 to Ala substantially diminishes, but does not eliminate, TGN to basolateral targeting. Mutation of other residues had little or no effect. The structure of a synthetic 17-residue peptide corresponding to this signal has been determined by two-dimensional nuclear magnetic resonance spectroscopy (4). This peptide tends to adopt a putative type 1 ␤-turn, encompassing residues 658 -661, followed by a nascent helical structure. In MDCK cells polarized sorting takes place in both the TGN and after endocytosis. Mutations that diminish TGN to basolateral sorting and increase TGN to apical sorting have a similar effect on sorting in the endocytotic pathway, decreasing recycling to the basolateral surface and...

“…Glutathione S-Transferase (GST) Fusion Proteins-GST fusion proteins of complexin 2 and mutants were purified by glutathione affinity chromatography and either left on the beads or released by thrombin cleavage as described previously (29). Single-point mutations in complexin 2 were made using QuikChange Lightning Site-directed Mutagenesis Kit (Stratagene).…”

Section: Methodsmentioning

confidence: 99%

Complexin 2 Modulates Vesicle-associated Membrane Protein (VAMP) 2-regulated Zymogen Granule Exocytosis in Pancreatic Acini

Falkowski

Thomas

Groblewski

2010

Self Cite

Complexins are soluble proteins that regulate the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion. Neuronal specific complexin 1 has inhibitory and stimulatory effects on exocytosis by clamping trans-SNARE complexes in a prefusion state and promoting conformational changes to facilitate membrane fusion following cell stimulation. Complexins are unable to bind to monomeric SNARE proteins but bind with high affinity to ternary SNARE complexes and with lower affinity to target SNARE complexes. Far less is understood about complexin function outside the nervous system. Pancreatic acini express the complexin 2 isoform by RT-PCR and immunoblotting. Immunofluorescence microscopy revealed complexin 2 localized along the apical plasma membrane consistent with a role in secretion. Accordingly, complexin 2 was found to interact with vesicle-associated membrane protein (VAMP) 2, syntaxins 3 and 4, but not with VAMP 8 or syntaxin 2. Introduction of recombinant complexin 2 into permeabilized acini inhibited Ca 2؉ -stimulated secretion in a concentration-dependent manner with a maximal inhibition of nearly 50%. Mutations of the central ␣-helical domain reduced complexin 2 SNARE binding and concurrently abolished its inhibitory activity. Surprisingly, mutation of arginine 59 to histidine within the central ␣-helical domain did not alter SNARE binding and moreover, augmented Ca 2؉ -stimulated secretion by 130% of control. Consistent with biochemical studies, complexin 2 colocalized with VAMP 2 along the apical plasma membrane following cholecystokinin-8 stimulation. These data demonstrate a functional role for complexin 2 outside the nervous system and indicate that it participates in the Ca 2؉ -sensitive regulatory pathway for zymogen granule exocytosis.Complexin 1, originally identified as synaphin, is a small cytosolic protein that associates with soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, which are essential to drive membrane fusion during neurotransmitter exocytosis (1, 2). Although complexin 1 is expressed exclusively in neurons, complexin 2 appears to be ubiquitously expressed and presumably functions in other secretory cell systems. Complexins 1 and 2 are highly homologous, each containing 134 amino acids that are 86% identical (3). Moreover, rat, mouse, and human complexin 2 proteins are 100% identical, although the nucleotide sequences of their mRNAs are considerably different (4). Recently, two other isoforms, complexins 3 and 4, were identified; however, evidence of their specific function is unclear. Complexin 3 is expressed strongly in retina and certain regions of the brain including the hippocampus and thalamus, whereas complexin 4 is only expressed in retina (5).In neurons, the SNARE complex is a four-helical bundle composed of vesicle associated membrane protein (VAMP) 2 2, and the target SNAREs (t-SNAREs), syntaxin 1 and SNAP 25, located on the plasma membrane (6). Complexin 1 was orig...