Isoproterenol-dependent redistribution of Rab27, Slac2-c and Slp4-a in parotid glands

Objective: Small GTPase Rab is a large family of putative membrane trafficking proteins. We have previously reported that Rab27 and its effectors, Slac2-c/MyRIP and Slp4-a/granuphilin-a, regulate isoproterenol (IPR)-stimulated amylase release from rat parotid acinar cells. However, nothing is known about their intracellular localization after β-stimulation. In the present study, we investigated the IPR-dependent redistribution of these proteins in rat parotid acinar cells.

Methods: The male Wistar rats (approximately 10 weeks old) parotid gland slices or the acinar cells were incubated with 1 µM IPR for 5 min. or 30 min., and were immediately put on ice for the next steps. Subcellular distribution of Rab27 and its effectors was analyzed by Western blotting and immunohistochemistry using specific antibodies. To detect degradation of Rab27, Slac2-c and Slp4-a, the subcellular fractions were prepared from the parotid homogenate which was incubated with or without 1 mM Ca²⁺ at 37ﾟC for 30 min, and then they were analyzed by Western blotting.

Results: Rab27A, Rab27B, Slac2-c and Slp4-a were detected in the secretory granule membrane fraction before stimulation. Immunofluorescence micrographs demonstrated that these proteins assembled into the apical domain after IPR-stimulation for 5 min, and that Rab27 and Slac2-c disappeared at 30 min. Western blotting also showed that Rab27 and Slac2-c were increased in the apical plasma membrane by 5 min-stimulation. After IPR-stimulation for 30 min Rab27 almost faded away from the apical plasma membrane, and Slac2-c disappeared in the cytoplasm fraction. Slac2-c protein in the cytoplasm was digested by incubation with Ca²⁺.

Conclusion: Our results suggest that rapid translocation of Rab27, Slac2-c and Slp4-a to the apical domain mediates IPR-stimulated amylase release. Subsequently, Rab27 fades away from the apical plasma membrane, and Slac2-c is likely to be degraded by Ca²⁺-dependent proteases in the cytosol.