Exophilin4/Slp2-a targets glucagon granules to the plasma membrane through unique Ca2+-inhibitory phospholipid-binding activity of the C2A domain - PubMed (original) (raw)
Exophilin4/Slp2-a targets glucagon granules to the plasma membrane through unique Ca2+-inhibitory phospholipid-binding activity of the C2A domain
Miao Yu et al. Mol Biol Cell. 2007 Feb.
Abstract
Rab27a and Rab27b have recently been recognized to play versatile roles in regulating the exocytosis of secretory granules and lysosome-related organelles by using multiple effector proteins. However, the precise roles of these effector proteins in particular cell types largely remain uncharacterized, except for those in pancreatic beta cells and in melanocytes. Here, we showed that one of the Rab27a/b effectors, exophilin4/Slp2-a, is specifically expressed in pancreatic alpha cells, in contrast to another effector, granuphilin, in beta cells. Like granuphilin toward insulin granules, exophilin4 promotes the targeting of glucagon granules to the plasma membrane. Although the interaction of granuphilin with syntaxin-1a is critical for the targeting activity, exophilin4 does this primarily through the affinity of its C2A domain toward the plasma membrane phospholipids phosphatidylserine and phosphatidylinositol-4,5-bisphosphate. Notably, the binding activity to phosphatidylserine is inhibited by a physiological range of the Ca(2+) concentration attained after secretagogue stimulation, which presents a striking contrast to the Ca(2+)-stimulatory activity of the C2A domain of synaptotagmin I. Analyses of the mutant suggested that this novel Ca(2+)-inhibitory phospholipid-binding activity not only mediates docking but also modulates the subsequent fusion of the secretory granules.
Figures
Figure 1.
Tissue and cell expression of exophilin4. (A) An equal amount of protein (35 μg) from tissue and cell extracts was loaded onto a polyacrylamide gel. Immunoblotting was performed using anti-exophilin4 antibodies. Numbers to the left of the panel are molecular masses in kilodaltons. (B) The pancreata of 16-wk-old male C3H/He mice were double immunostained with anti-exophilin4 and anti-glucagon antibodies. Merged fluorescent signals are also shown. Bar, 20 μm.
Figure 2.
Complex formation between exophilin4 and Rab27a. (A) Aliquots of glutathione beads containing either GST alone or GST-fused Rab27a preloaded with GDP or GTPγS (1 μg of protein) were incubated with αTC1.6 cell extracts (1 mg of protein) and then washed three times. Proteins that bound to the GST fusion proteins and an aliquot of the original cell lysates (20 μg of protein) were analyzed by immunoblotting with anti-exophilin4 antibodies. (B) Extracts of αTC1.6 cells (1 mg of protein) were incubated with anti-Rab27a, anti-Rab3a antibodies, or control mouse IgG and then with protein G-Sepharose beads. After washing the beads, immunoprecipitates (IP) and an aliquot of the original cell lysates (20 μg of protein) were analyzed by immunoblotting using anti-exophilin4 and anti-Rabs antibodies. Numbers to the left of each panel are molecular masses in kilodaltons.
Figure 3.
Intracellular distribution of exophilin4, Rab27a, and glucagon granules. (A) αTC1.6.6 cells were transfected with a plasmid encoding either HA-tagged exophilin4 (a–c) or Xpress-tagged Rab27a (d–f). They were then double-immunostained with anti-glucagon and either anti-HA (a–c) or anti-Xpress antibodies (d–f), and observed with a confocal microscope. Merged fluorescent signals are shown in c and f. (B) αTC1.6/phogrin-EGFP cells (a–c) or those transiently transfected with an expression plasmid encoding HA-tagged full-length (d–f) or C-terminal exophilin4 (579-910 amino acids; g–i) were fixed and stained with anti-glucagon (a–c) or anti-HA antibodies (d–i). Intrinsic EGFP fluorescence signals (a, d, and g), immunostaining signals (b, e, and h), and merged signals (c, f, and i) are shown. Bars, 10 μm.
Figure 4.
Subcellular localization of N-terminal and C-terminal portions of exophilin4. αTC1.6 cells were transfected with an expression plasmid encoding the full-length (a), N terminus (b), C2AB domain (c), C2A domain (d), or C2B domain (e) of exophilin4 with the HA-tag. The HA-tagged C2A domain harboring the DN (f) or KQ mutation (g) was similarly expressed in αTC1.6 cells. The cells were fixed and stained with anti-HA antibodies and observed with a confocal microscope. Bar, 10 μm.
Figure 5.
Liposome binding to the C2 domains of synaptotagmin I, granuphilin, and exophilin4. (A) Recombinant proteins were produced in E. coli and purified with glutathione-Sepharose 4B. Equivalent amounts (2 μg) of each GST-fused protein were electrophoresed on a 10% polyacrylamide gel and stained with Coomassie blue: GST protein (lane 1); synaptotagmin I-C2A (lane 2) and synaptotagmin-C2B (lane 3); exophilin4-C2A (lane 4), exophilin4-C2B (lane 5), and exophilin4-C2AB (lane 6); and granuphilin-C2A (lane 7), granuphilin-C2B (lane 8), and granuphilin-C2AB (lane 9). Numbers to the left of the panel are molecular masses in kilodaltons. (B) GST-fused recombinant proteins bound to glutathione-Sepharose 4B were incubated with 3H-labeled liposomes in the absence (open bars) or presence (black bars) of 100 μM Ca2+. 3H-labeled liposomes were composed of PC mixed with either PS (top) or PIP2 (bottom). Phospholipid binding was measured by scintillation counting of the beads after extensive washing and are represented as the means ± SEM (n = 7). (C) Binding of 3H-labeled liposomes containing PS to synaptotagmin I-C2A (circles) or exophilin4-C2A (squares) was examined in buffers containing various free Ca2+ concentrations. The data are normalized to the binding of synaptotagmin I-C2A at 100 μM Ca2+ or that of exophilin4-C2A without the addition of the CaCl2 solution, respectively, and are represented as means ± SEM (n = 3).
Figure 6.
Liposome binding to the wild-type and mutant C2A domains of exophilin4. (A) Equivalent amounts (2 μg) of GST protein (lane 1), wild type (lane 2), DN mutant (lane 3), and KQ mutant, GST-fused exophilin4-C2A proteins (lane 4) were electrophoresed and stained by Coomassie blue. Numbers to the left of the panel are molecular masses in kilodaltons. (B and C) GST-fused recombinant proteins were incubated with 3H-labeled liposomes containing either PS (B) or PIP2 (C) in the absence (open bars) or presence (black bars) of 100 μM Ca2+. Data are shown as means ± SEM (B, n = 7; C, n = 6).
Figure 7.
Granule-targeting activity of wild-type and mutant exophilin4. (A) αTC1.6/phogrin-EGFP cells were infected with adenovirus bearing β-gal, HA-tagged exophilin4WT, exophilin4DN, or exophilin4KQ cDNA. The expression levels of endogenous (arrow) and exogenous exophilin4 (arrowhead) were determined by immunoblotting with anti-exophilin4 antibodies (top). Exogenous human exophilin4 migrated slightly faster than endogenous mouse exophilin4. The expression levels of exogenous exophilin4 (arrowhead) were also examined by immunoblotting with anti-HA antibodies (bottom). (B) αTC1.6/phogrin-EGFP cells were infected with recombinant adenovirus encoding HA-tagged exophilin4WT (a–c), exophilin4DN (d–f), or exophilin4KQ (g–i), as well as control adenovirus encoding β-gal (j), under the same conditions as for A. The cells were fixed and stained with anti-HA and Cy3-labeled anti-rat IgG antibodies. Cy3 fluorescence (a, d, and g), intrinsic EGFP fluorescence (b, e, h, and j), and merged fluorescence (c, f, and i) are shown. Bar, 10 μm. (C) The targeting activities of wild-type and mutant exophilin4 examined as in B were quantified as described in Materials and Methods. Data are shown as means ± SEM (n = 6).
Figure 8.
Effect of overexpression of wild-type and mutant exophilin4 on glucagon and insulin secretion. (A) αTC1.6 and MIN6 cells were infected with adenoviruses bearing either β-gal or wild-type human exophilin4 cDNA. The expression levels of endogenous and exogenous exophilin4 were determined by immunoblotting with anti-exophilin4 antibodies (top). Exogenous human exophilin4 (arrowhead) migrates slightly faster than endogenous mouse exophilin4 (small arrow). Note that MIN6 cells do not express exophilin4 endogenously. The infected αTC1.6 cells were incubated for 30 min in either modified KRB (16.7 mM glucose; open bars) or the buffer modified to include high K+ (60 mM KCl and 1.38 mM glucose; solid bars). The infected MIN6 cells were incubated for 30 min in either modified KRB (including 1.38 mM glucose; open bars) or the buffer modified to include high K+ (60 mM KCl and 16.7 mM glucose; solid bars). Secreted glucagon or insulin in the media was measured (bottom). Note that exogenous expression of exophilin 4 does not affect evoked insulin secretion in MIN6 cells, in contrast to the inhibition of glucagon secretion in αTC1.6 cells. (B) αTC1.6 cells infected by adenovirus as in Figure 7A and the effect of expression of wild-type and mutant exophilin4 on glucagon secretion was examined as in A. Values are normalized to the release of glucagon or insulin from uninfected cells stimulated by high K+ and are given as means ± SEM. *p < 0.001 versus high K+-stimulated cells infected with the same titer of the virus bearing β-gal cDNA (A and B, n = 6).
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