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. 2003 Nov 19;23(33):10531-9.
doi: 10.1523/JNEUROSCI.23-33-10531.2003.

Divergent functions of neuronal Rab11b in Ca2+-regulated versus constitutive exocytosis

Mikhail V Khvotchev  1 , Mindong Ren, Shigeo Takamori, Reinhard Jahn, Thomas C Südhof
Affiliations

Divergent functions of neuronal Rab11b in Ca2+-regulated versus constitutive exocytosis

Mikhail V Khvotchev et al. J Neurosci. .

Abstract

Using PC12 cells that express transfected human growth hormone (hGH) as a secreted reporter protein, we have searched for Rab proteins that function in exocytosis. Among the Rab proteins tested, we found that besides the previously described Rab3 proteins, only members of the Rab11 family (Rab11a, 11b, and 25) impaired Ca2+-induced exocytosis. Rab11b, which is enriched in brain, had the strongest effect. Consistent with a role in exocytosis, Rab11 and Rab3 proteins were colocalized with other vesicle proteins on secretory vesicles in PC12 cells and on mature synaptic vesicles in brain. Rab11b mutants that fix Rab11b in the GTP- or GDP-bound state both effectively inhibited Ca2+-induced exocytosis but seemed to act by distinct mechanisms: whereas GDP-bound Rab11b greatly stimulated constitutive secretion of hGH and depleted hGH stores in secretory vesicles, GTP-bound Rab11b only had a moderate effect on constitutive secretion and no effect on vesicular hGH stores. These results suggest that, consistent with a GTP-dependent regulation of Rab function, GDP-bound Rab11b indirectly inhibits Ca2+-triggered exocytosis by causing the loss of hGH from the PC12 cells, whereas GTP-bound Rab11b directly impairs Ca2+-triggered exocytosis. In contrast to neuroendocrine PC12 cells in which GTP- and GDP-bound Rab11b inhibited Ca2+-induced, but not constitutive, exocytosis, in non-neuronal cells GTP- and GDP-bound Rab11b inhibited constitutive exocytosis and caused an accumulation of cellular hGH. Viewed together, our data suggest that, in addition to other functions, Rab11 has a specific role in neuronal and neuroendocrine but not in non-neuronal cells as a GTP-dependent switch between regulated and constitutive secretory pathways.

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Figures

Figure 1.
Figure 1.
Rab11b inhibits regulated exocytosis in PC12 cells. A, Specificity of the inhibitory effect of Rab11b on regulated exocytosis. PC12 cells were cotransfected with an hGH reporter vector and various expression vectors: a control vector encoding no protein or vectors encoding the Rab proteins that are identified by their number below the bar diagrams. Seventy-two hours after transfection, cells were stimulated with control medium (□) or with depolarizing medium containing 56 mm KCl (▪) for 15 min, and the amount of hGH released into the medium and retained in the cells was determined by RIA. hGH release was calculated as the fraction of total hGH and normalized for the K+-induced secretion obtained under control conditions (100%). Results shown are means ± SEMs from four independent experiments performed in duplicates. B, Expression of GFP-fused Rab11/25 proteins in PC12 cells. PC12 cells were cotransfected with an hGH expression vector and either an expression vector encoding GFP (control) or GFP-fused wild-type Rab11a, Rab11b, and Rab25 proteins. The expression of the recombinant and endogenous Rab11/25 proteins was verified by immunoblotting. C, Effects of transfected GFP-Rab11/25 proteins on regulated exocytosis tested with the hGH cotransfection assay. The results were normalized for the K+-induced secretion obtained under control conditions (100%). Data are from three independent experiments performed in duplicates.
Figure 2.
Figure 2.
Inhibition of regulated exocytosis by Rab11b is independent of the secretagogue and the GTP/GDP-binding or prenylation status of Rab11b. PC12 cells were cotransfected with an hGH expression vector and either a control expression vector (C) or expression vectors encoding wild-type (WT) and mutant Rab11b proteins (see Results). Cells were treated under control conditions (□) or stimulated by K+ depolarization (KCl; ▪) or 0.5 nm α-latrotoxin (Ltx;▦). Secretion was calculated as described above, except that the results were not normalized to the control condition. Data were derived from a single representative experiment performed in duplicates and repeated three times.
Figure 3.
Figure 3.
GFP-fused Rab11b proteins are potent inhibitors of K+-induced exocytosis in PC12 cells. A, PC12 cells were cotransfected with an hGH expression vector and either an expression vector encoding GFP (control) or GFP-fused wild-type and mutant Rab11b proteins. The expression of the recombinant and endogenous Rab11b proteins was verified by immunoblotting. B, Effects of transfected GFP-Rab11b proteins on regulated exocytosis tested with the hGH cotransfection assay. Data were derived from a single representative experiment performed in duplicates and repeated three times. C, Localization of GFP-fused Rab11b proteins (a-d) determined by confocal laser microscopy. The position of nucleus is indicated by N. Scale bar, 2 μm.
Figure 4.
Figure 4.
Characterization of Rab11 antibodies. A, COS cells were transfected with an empty vector (control) or Rab3a-d, Rab5a, Rab8a, Rab11b, or Rab13 expression vectors. Two days after transfection, cellular proteins were analyzed by immunoblotting using a newly generated polyclonal antibody against Rab11b or monoclonal antibodies against Rab3, Rab5, or the hemagglutinin antigen (HA) tag that was fused to Rab8a, 11b, and 13. Additional lower bands are attributable to incomplete geranylgeranylation of Rab proteins in transfected COS cells (Johnston et al., 1991). B, COS cells were transfected with an expression vector encoding GFP (control) or GFP-fused Rab11a, Rab11b, and Rab25. Two days after transfection, cellular proteins were analyzed by immunoblotting using antibody against GFP and polyclonal and monoclonal antibodies against Rab11 proteins.
Figure 5.
Figure 5.
Subcellular fractionation of PC12 cells: identification of secretory compartments. PC12 cells were transfected with hGH as described above. Two days after transfection, PC12 cells were labeled overnight with 3H-norepinephrine and lysed, and organelles in the postnuclear supernatants were separated on discontinuous sucrose gradients. Gradient fractions were analyzed by liquid scintillation counting to measure 3H-norepinephrine (•, top), by RIA to determine hGH (○, top) and by immunoblotting of membranes for synaptophysin 1 (Syp 1), syntaxin 1, Rab3a, Rab11, GDIα, Munc18, and Rab5a (bottom).
Figure 6.
Figure 6.
Rab11 is enriched on purified synaptic vesicles. Immunoblot analysis of fractions obtained during the purification of synaptic vesicles by differential centrifugation and controlled pore-glass chromatography. Note that Rab11 exhibits co-enrichment with synaptic vesicle markers such as synaptophysin 1 (Syp 1), synaptotagmin 1 (Syt 1), and Rab3a but not with plasma membrane proteins SNAP-25, glutamate receptors GluR1and NMDA-R, or with soluble cytosolic protein α-SNAP. The purification procedure was repeated two times with identical results.
Figure 7.
Figure 7.
Effect of Rab11b proteins on constitutive exocytosis in PC12 cells. PC12 cells were cotransfected with expression vectors encoding hGH and wild-type or mutant Rab11b. Two GTPase-deficient Rab11b mutants were used: S20V (GTPa) and Q70L (GTPb). hGH secreted into the extracellular medium was measured by RIA on days 2-4 after transfection and is calculated as a fraction of the hGH remained in the cells and normalized to the value for control transfected cells. Data shown are means ± SEMs from five independent experiments performed in triplicates.
Figure 8.
Figure 8.
Large dense core vesicles storage is depleted in PC12 cells transfected with GDP-bound Rab11b. PC12 cells were cotransfected expression vectors encoding hGH and wild-type Rab11b or GDP-bound mutant Rab11b. Subcellular fractionation was performed as described in Figure 5. Gradient fractions were analyzed for hGH by RIA.
Figure 9.
Figure 9.
Effect of Rab11/25 proteins on constitutive exocytosis in HEK293 cells. HEK293 cells were cotransfected with expression vectors encoding hGH and GFP alone (control) or GFP-fused Rab11a, Rab11b, and Rab25 proteins. hGH was measured in the cells and aliquots of the medium 2 days after transfection. Secreted hGH (A, ▪) and remaining cellular hGH (B, ▦) were calculated as described for Figure 7. Data shown are means ± SEMs from three independent experiments performed in duplicates.
Figure 10.
Figure 10.
Effect of wild-type and mutant Rab11b proteins on constitutive exocytosis in HEK293 and HeLa cells. HEK293 (A) and HeLa (B) cells were cotransfected with expression vectors encoding hGH and no insert (control) or various Rab11b proteins. hGH was measured in the cells and aliquots of the medium 2 days after transfection. Secreted hGH (left, ▪) and remaining cellular hGH (right, [gbox) were calculated as described for Figure 7. Data shown are means ± SEMs from three independent experiments performed in triplicates.
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