Roles of Munc18-3 in amylase release from rat parotid acinar cells

Several "soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor" (SNARE) proteins have been identified in rat parotid acinar cells, including VAMP-2, syntaxin 4, and SNAP-23. Furthermore, an association between Munc18c (Munc18-3) and syntaxin 4 has been reported. However, the role of Munc18-3 in secretory granule exocytosis on parotid acinar cells remains unclear. In the present study, we investigated the role of Munc18-3 in rat parotid acinar cells. Munc18-3 was localized on the apical plasma membrane where exocytosis occurs and interacted with syntaxin 4. Anti-Munc18-3 antibody dose-dependently decreased isoproterenol (IPR)-induced amylase release from SLO-permeabilized parotid acinar cells. Furthermore, stimulation of the acinar cells with IPR induced translocation of Munc18-3 from the plasma membrane to the cytosol. Munc-18-3 was not phosphorylated by a catalytic subunit of protein kinase (PK) A but phosphorylated by PKC. Treatment of the plasma membrane with PKC but not PKA induced displacement of Munc18-3 from the membrane. The results indicate that Munc18-3 regulates exocytosis in the acinar cells for IPR-induced amylase release and that phosphorylation of Munc18-3 by PKA is not involved in the mechanism.     

The cyclic nucleotide specificity of eight cAMP-binding proteins in Dictyostelium discoideum is correlated into three groups

cAMP is a mediator of inter- and intracellular events in Dictyostelium discoideum and is thought to act through specific receptors. Eight forms of cAMP-binding proteins have been described in this organism: four forms of a cell surface receptor, a cell surface and extracellular phosphodiesterase, an intracellular cAMP-dependent protein kinase (CAK), and a recently identified cAMP-binding protein (CABP1) that is present on the cell surface, in the cytoplasm, and in the nucleus. In this study we have analyzed the cyclic nucleotide specificity of these cAMP-binding proteins using 13 derivatives of cAMP with modifications in the adenine, ribose, and phosphate moiety. The results suggest that the cAMP-binding proteins belong to three groups: (i) four forms of the cell surface receptor, (ii) two forms of an intracellular receptor (CABP1 and CAK), and (iii) cell surface and extracellular phosphodiesterase. cAMP is probably bound to the surface receptors in the anti conformation in a hydrophobic cleft of the receptor with essential interactions at N6H2' and O3'. In contrast, cAMP is probably bound to CAK and CABP1 in the syn conformation with essential interactions at O2', O3', O5', and exocyclic oxygen. Finally, binding of cAMP to phosphodiesterase involves only O3' and exocyclic oxygen. The cyclic nucleotide specificity of cAMP-induced processes in D. discoideum indicates that the cell surface receptors participate in the transduction of the cAMP signal during chemotaxis and cell differentiation. Functions for CABP1 and CAK in these processes are presently elusive.     

Function of plastid mRNA 3' inverted repeats. RNA stabilization and gene-specific protein binding

Plastid protein coding regions in plants are generally flanked by 3' inverted repeat (IR) sequences. In a previous work (Stern, D. B., and Gruissem, W. (1987) Cell 51, 1145-1157), we have shown that their role may be in RNA stabilization and as a processing signal that establishes the mature mRNA 3' end. In this report we have investigated the stability and protein interaction of chloroplast mRNA 3' IR-RNA sequences in more detail. Progressive deletions into the 3' IR-RNA sequences for the chloroplast cytochrome b6/f subunit IV (petD) mRNA reduce the stability of the RNA, indicating that the potential to form a stem/loop is a minimum requirement for petD 3' IR-RNA stability in vitro. Specific point mutants also destabilize the processed 3' IR-RNA, suggesting an important role for the primary sequence. Gel mobility shift and UV-cross-linking analysis has shown that 3' IR-RNAs of petD and two other chloroplast mRNAs (rbcL and psbA) interact with proteins in vitro. Comparison of the bound petD 3' IR-RNA proteins with proteins that bind to rbcL and psbA reveals that binding of certain proteins is gene-specific. Also, precursor and processed petD 3' IR-RNAs bind different sets of proteins. A single nucleotide transversion (T----A) near the base of the stem eliminates the binding of a 29-kDa protein to the petD 3' IR-RNA precursor. We discuss the possible role of 3' IR-RNA-protein interactions in plastid mRNA 3' end maturation and differential mRNA stability.     

Transcriptional activity in previtellogenic oocyte germinal vesicles from Xenopus laevis

Receptor interactions of parotid acinar cells with beta-agonists are mediated by cyclic 3',5'-monophosphate (cAMP) and expressed as cAMP-dependent protein kinase (cAPK) activation. In addition to its location in the cytoplasm, we have shown that cAPK is associated with the nuclear non-histone protein (NHP) fraction (0.35 M NaCl extract) of rat parotid acinar cells. Nuclei were prepared from isolated parotid acini with minimal contamination from other cell types or cytoplasmic components. The nuclear cAPK activity was inhibited by the thermostable inhibitor and was stimulated by the addition of exogenous cAMP to the assay, indicating that the enzyme is present in the holoenzyme form. Enzyme activity was not increased in the presence of detergent, suggesting that cAPK is not bound to the nuclear membrane. Photoaffinity-labeling studies with an 8-azido analog of cAMP showed that regulatory subunits of both type I and type II cAPK isozymes are present in parotid cell nuclei. Short-term in vitro stimulation of the acini with 10(-6) M isoproterenol did not alter cAPK activity in the nuclear fraction. These findings indicate that compartmentation of cAPK into nuclear and extranuclear locations in rat parotid acinar cells is similar to that of several other cell types which are responsive to hormonal stimulation.     

Immunocytochemical localization of a developmentally regulated, isoproterenol-inducible protein (LM protein) in rat submandibular gland

Administration of the beta-adrenergic drug isoproterenol (IPR) produces hyperplastic and hypertrophic enlargements of the submandibular gland of the rat and induces the synthesis of specific proteins in this organ. One of these proteins, the LM (large mobile) protein, was demonstrated immunocytochemically in the submandibular glands of developing untreated and IPR-treated rats. Immunoreactive LM protein was absent in the glands of 20-day-old fetuses and 1- and 2-day-old rats. It was localized in the proacinar and immature acinar cells in the glands of 6- to 21-day-old animals, but it was undetectable at 28 days of age. In the glands of adult rats, secretory granules of the granular convoluted tubule cells showed immunostaining for the LM protein which was also present in trace amounts in the acinar cells. Daily administration of IPR for 5 days to newborn or 8- or 15-day-old rats caused an apparent acceleration of proacinar/acinar cell differentiation, and consequently it increased the frequency of cells immunostained for the LM protein as well as the amount of immunoreactive material in these cells. Thus, the expression of LM protein in the submandibular gland is developmentally regulated, and it is restricted to the stage of differentiation of proacinar cells from terminal tubule cells. IPR is capable of inducing this protein in fully differentiated acinar cells in 3-week-old or older animals.     

Phosphorylation of P-Rex1 by the cyclic AMP-dependent protein kinase inhibits the phosphatidylinositiol (3,4,5)-trisphosphate and Gbetagamma-mediated regulation of its activity

Rac activation is a key step in chemotaxis of hematopoietic cells, which is both positively and negatively regulated by receptors coupled to heterotrimeric G proteins. P-Rex1, a Rac-specific guanine nucleotide exchange factor, is dually activated by phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)) and the Gbetagamma subunits of heterotrimeric G proteins. This study explored the regulation of P-Rex1 by phosphorylation with the cAMP-dependent protein kinase (protein kinase A) in vitro and by G(i)- and G(s)-coupled receptors in HEK293T cells. P-Rex1 isolated from Sf9 and HEK293T cells migrates as two distinct bands that are partially phosphorylated. Phosphorylation of P-Rex1 with protein kinase A (PKA) inhibits the PIP(3)- and Gbetagamma-stimulated P-Rex1 guanine nucleotide exchange activity on Rac. The guanine nucleotide exchange factor activity of three different forms of P-Rex1 (native Sf9, de-phosphorylated, and phosphorylated) was examined in the presence of PIP(3) and varying concentrations of Gbeta(1)gamma(2). Gbeta(1)gamma(2) was 47-fold less potent in activating the phosphorylated form of P-Rex1 compared with the de-phosphorylated form. HEK293T cells expressing P-Rex1 were labeled with (32)P and stimulated with lysophosphatidic acid (LPA) to release Gbetagamma or isoproterenol to activate PKA. Treatment with isoproterenol or S(p)-cAMPS, a potent activator of PKA, increased the incorporation of (32)P into P-Rex1. LPA increased the amount of GTP-bound Rac in the cells and isoproterenol reduced basal levels of GTP-bound Rac and blunted the effect of LPA. Treatment of the cells with S(p)-cAMPS also reduced the levels of GTP-bound Rac. These results outline a novel mechanism for G(s)-linked receptors to regulate the function of P-Rex1 and inhibit its function in cells.     

Nucleotide-dependent conformational changes in the sigma54-dependent activator DctD

Activators of sigma(54)-RNA polymerase holoenzyme couple ATP hydrolysis to formation of an open promoter complex. DctD(Delta1-142), a truncated and constitutively active form of the sigma(54)-dependent activator DctD from Sinorhizobium meliloti, displayed an altered DNase I footprint at its binding site located upstream of the dctA promoter in the presence of ATP. The altered footprint was not observed for a mutant protein with a substitution at or near the putative arginine finger, a conserved arginine residue thought to contact the nucleotide. These data suggest that structural changes in DctD(Delta1-142) during ATP hydrolysis can be detected by alterations in the DNase I footprint of the protein and may be communicated by interactions between bound nucleotide and the arginine finger. In addition, kinetic data for changes in fluorescence energy transfer upon binding of 2'(3')-O-(N-methylanthraniloyl)-ATP (Mant-ATP) to DctD(Delta1-142) and DctD suggested that these proteins undergo multiple conformational changes following ATP binding.     

A new member of the glutamine-rich protein gene family is characterized by the absence of internal repeats and the androgen control of its expression in the submandibular gland of rats

A cDNA, corresponding to a rat submandibular mRNA which is accumulated at a 20-fold higher level in males than females, has been isolated. The predicted protein, SMR2, has a calculated molecular mass of 15.4 kDa and is rich in glutamine/glutamic acid, proline, and asparagine/aspartic acid, a characteristic of the so-called salivary glutamine-rich proteins (GRPs) of the submandibular gland of rats. Nucleotide sequence comparisons indeed revealed strong similarities between the sequences of the SMR2 mRNA and that of GRPs, except in the region encoding the carboxyl-terminal part of the proteins. In particular, the SMR2 mRNA contains the 5'-untranslated region and the signal peptide region shared by both groups of GRPs and proline-rich proteins (PRPs). A major difference is that, in SMR2, the peptidic motif which is repeated four or five times in GRPs, is only found once. The SMR2 gene is about 3.5 kilobases in length and contains 4 exons. The second intron, which does not exist in characterized GRP genes, splits the "transition" region which separates the repetitive sequences from the signal peptide. This structure is reminiscent of that found in most PRP genes, strengthening the hypothesis that GRP and PRP genes have the same ancestral origin.