The Rab27a/Granuphilin Complex Regulates the Exocytosis of Insulin-Containing Dense-Core Granules

Abstract

Recently, we identified and characterized a novel protein, granuphilin, whose domain structure is similar to that of the Rab3 effector protein rabphilin3 (J. Wang, T. Takeuchi, H. Yokota, and T. Izumi, J. Biol. Chem. 274:28542-28548, 1999). Screening its possible Rab partner by a yeast two-hybrid system revealed that an amino-terminal zinc-finger domain of granuphilin interacts with Rab27a. Granuphilin preferentially bound to the GTP form of Rab27a. Formation of the Rab27a/granuphilin complex was readily detected in the pancreatic beta cell line MIN6. Moreover, the tissue distributions of Rab27a and granuphilin are remarkably similar: both had significant and specific expression in pancreatic islets and in pituitary tissue, but no expression was noted in the brain. Analyses by immunofluorescence, immunoelectron microscopy, and sucrose density gradient subcellular fractionation showed that Rab27a and granuphilin are localized on the membrane of insulin granules. These findings suggest that granuphilin functions as a Rab27a effector protein in beta cells. Overexpression of wild-type Rab27a and its GTPase-deficient mutant significantly enhanced high K⁺-induced insulin secretion without affecting basal insulin release. Although Rab3a, another exocytotic Rab protein, has some similarities with Rab27a in primary sequence, intracellular distribution, and affinity toward granuphilin, overexpression of Rab3a caused different effects on insulin secretion. These results indicate that Rab27a is involved in the regulated exocytosis of conventional dense-core granules possibly through the interaction with granuphilin, in addition to its recently identified role in lysosome-related organelles.

This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan. It was also supported in part by the Ministry of Health and Welfare of Japan (Research on Human Genome and Gene Therapy) and by grants from the Japan Diabetes Foundation, the Suzuken Memorial Foundation, and the Japan Insulin Study Group Award, to T.I.

We thank S. M. Hollenberg (Vollum Institute, Oregon Health Sciences University) for providing reagents of the yeast two-hybrid system and H. Fujita (Kyushu University) for providing melanocyte cell lines. We also thank E. Erikson (University of Colorado School of Medicine) for critical reading of the manuscript and H. Tamemoto, S. Mizutani, J. Wang, and K. Nagashima (Institute for Molecular and Cellular Regulation, Gunma University) for generous support and useful advice.