Insulin release from pancreatic ␤-cells plays an essential role in blood glucose homeostasis. Several proteins controlling insulin exocytosis have been identified, but the factors determining the expression of the components of the secretory machinery of ␤-cells remain largely unknown. MicroRNAs are newly discovered small non-coding RNAs acting as repressors of gene expression. We found that overexpression of mir-9 in insulinsecreting cells causes a reduction in exocytosis elicited by glucose or potassium. We show that mir-9 acts by diminishing the expression of the transcription factor Onecut-2 and, in turn, by increasing the level of Granuphilin/Slp4, a Rab GTPase effector associated with ␤-cell secretory granules that exerts a negative control on insulin release. Indeed, electrophoretic mobility shift assays, chromatin immunoprecipitation, and transfection experiments demonstrated that Onecut-2 is able to bind to the granuphilin promoter and to repress its transcriptional activity. Moreover, we show that silencing of Onecut-2 by RNA interference increases Granuphilin expression and mimics the effect of mir-9 on stimulus-induced exocytosis. Our data provide evidence that in insulin-producing cells adequate levels of mir-9 are mandatory for maintaining appropriate Granuphilin levels and optimal secretory capacity.The insulin in the circulation of adult mammals is produced exclusively by pancreatic ␤-cells, making them the central regulators of glucose homeostasis. Alterations in ␤-cell secretory function can cause hyperglycemia and lead to diabetes mellitus. During the last few years a number of key components of the machinery controlling insulin exocytosis have been identified. These components include different SNARE 3 proteins, Rab3 and Rab27 GTPases, and their effectors Noc2 and Granuphilin/ Slp4 (1-3). Alteration in the function or in the expression level of these proteins results in insulin secretory defects both in vitro (4 -8) and in vivo (9 -11). Despite recent progress in understanding the molecular mechanisms regulating the final events in the secretory pathway of ␤-cells, the factors determining the expression of the proteins involved in insulin exocytosis are largely unknown.MicroRNAs (miRNAs) are newly discovered regulators of gene expression that act by targeting the 3Ј-untranslated region (3Ј-UTR) of mRNA sequences and by preventing the productive translation of the messengers (12-15). miRNAs have been implicated in many processes in invertebrates, including cell proliferation, apoptosis (16 -18), fat metabolism (17), and neuronal patterning (19). Because of their spatial and temporal expression pattern and their conservation across species (20 -22), miRNAs are believed to play similar roles in all animal cells. In mammals, only for a few miRNAs a specific function has been assigned. A subset of miRNAs has been shown to be involved in metabolic regulation: mir-143 participates in human adipocyte differentiation (23), and the levels of mir-375, a pancreatic islet-specific miRNA, influence insulin...

To define the role of the Rab3-interacting molecule RIM in exocytosis we searched for additional binding partners of the protein. We found that the two C 2 domains of RIM display properties analogous to those of the C 2 B domain of synaptotagmin-I. Thus, RIM-C 2 A and RIM-C 2 B bind in a Ca 2؉ -independent manner to ␣1B, the pore-forming subunit of N-type Ca 2؉ channels (EC 50 ‫؍‬ ϳ20 nM). They also weakly interact with the ␣1C but not the ␣1D subunit of L-type Ca 2؉ channels. In addition, the C 2 domains of RIM associate with SNAP-25 and synaptotagmin-I. The binding affinities for these two proteins are 203 and 24 nM, respectively, for RIM-C 2 A and 224 and 16 nM for RIM-C 2 B. The interactions of the C 2 domains of RIM with SNAP-25 and synaptotagmin-I are modulated by Ca 2؉ . Thus, in the presence of Ca 2؉ (EC 50 ‫؍‬ ϳ75 M) the interaction with synaptotagmin-I is increased, whereas SNAP-25 binding is reduced. Synaptotagmin-I binding is abolished by mutations in two positively charged amino acids in the C 2 domains of RIM and by the addition of inositol polyphosphates. We propose that the Rab3 effector RIM is a scaffold protein that participates through its multiple binding partners in the docking and fusion of secretory vesicles at the release sites.Secretion of neurotransmitters, polypeptide hormones, and a variety of other proteins occurs by exocytosis, a multistage process including targeting, docking, and, finally, fusion of secretory vesicles with the plasma membrane. During the last few years, a combination of genetic and biochemical approaches has lead to the identification of several proteins involved in this complex cascade of events. Most of these proteins turned out to be specialized components of the evolutionary conserved machinery that governs intracellular vesicular trafficking in eukaryotic cells (1). Exocytosis was found to necessitate the assembly of a ternary complex between the vesicular SNARE 1 VAMP, associated with the secretory vesicle, and the target SNAREs syntaxin-1 and SNAP-25, localized at the plasma membrane (2). The SNARE complex was initially proposed to ensure the docking of secretory vesicles at the plasma membrane (3). However, it is unlikely that SNARE assembly constitutes the sole determinant for the targeting of secretory vesicles at the release sites because SNARE pairing is rather promiscuous (4, 5), and the localization of syntaxin-1 and SNAP-25 is not restricted to active zones (6). A current hypothesis, supported by biochemical and structural data, proposes that the assembly of the heterotrimeric complex between VAMP, SNAP-25, and syntaxin-1 provides the driving force for membrane fusion (7).In most secretory systems, the exocytotic process is initiated by an increase in the intracellular Ca 2ϩ concentration. In some cells, such as neurons, the elevation of Ca 2ϩ ions is due to opening of voltage-gated calcium channels that are clustered at the release sites, whereas in others, Ca 2ϩ ions are mobilized from intracellular stores. Biochemical and genetic studies indicate...

Granuphilin/Slp-4 is a member of the synaptotagmin-like protein family expressed in pancreatic beta-cells and in the pituitary gland. We show by confocal microscopy that both granuphilin-a and -b colocalize with insulin-containing secretory granules positioned at the periphery of pancreatic beta-cells. Overexpression of granuphilins in insulin-secreting cell lines caused a profound inhibition of stimulus-induced exocytosis. Granuphilins were found to bind to two components of the secretory machinery of pancreatic beta-cells, the small GTP-binding protein Rab3 and the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-binding protein Munc-18. The interaction with Rab3 occurred only with the GTP-bound form of the protein and was prevented by a point mutation in the effector domain of the GTPase. Structure-function studies using granuphilin-b mutants revealed that complete loss of Rab3 binding is associated with a reduction in the capacity to inhibit exocytosis. However, the granuphilin/Rab3 complex alone is not sufficient to mediate the decrease of exocytosis, suggesting the existence of additional binding partners. Taken together, our observations indicate that granuphilins play an important role in pancreatic beta-cell exocytosis. In view of the postulated role of Munc-18 in secretory vesicle docking, our data suggest that granuphilins may also be involved in this process.

Rab GTPases regulate membrane traffic between the cellular compartments of eukaryotic cells. Rab3 is associated with secretory vesicles of neuronal and endocrine cells and controls the Ca 2⍣ -triggered release of neurotransmitters and hormones. To clarify the mode of action of Rab3 we generated mutants of the GTPase that do not interact efficiently with its putative effectors Rabphilin and RIM. Surprisingly, these mutants transfected in PC12 cells were still capable of inhibiting Ca 2⍣ -evoked secretion. Rab3 was shown previously to bind to calmodulin in a Ca 2⍣ -dependent manner. By replacing two arginines conserved between Rab3 isoforms, we generated a mutant with a reduced affinity for calmodulin. This mutant retained the capacity to interact with the Rab3 regulatory proteins, Rabphilin, RIM, Mss4 and RabGDI, and was correctly targeted to dense-core secretory granules. However, replacement of the two arginines abolished the ability of the GTP-bound form of Rab3 to inhibit exocytosis of catecholamine-and insulin-secreting cells. We propose that a Rab3-calmodulin complex generated by elevated Ca 2⍣ concentrations mediated at least some of the effects of the GTPase and limited the number of exocytotic events that occurred in response to secretory stimuli.

Rab3 GTPases regulate exocytosis of neurons, endocrine and exocrine cells. In the present paper, we report a system to measure the guanine nucleotide status of Rab3 proteins in living cells. The assay is based on the ability of the Rab3 interacting molecule RIM to extract selectively the GTP-bound form of Rab3. Using this system, we found that approx. 20% of wild-type Rab3A, -B, -C or -D transfected in the insulin-secreting cell line HIT-T15 is in the GTP-bound conformation. The pool of activated Rab3 is decreased under conditions that stimulate exocytosis or by co-expression of the Rab3 GTPase-activating protein. In contrast, co-expression of Mss4 or Rab3-GEP (guanine nucleotide exchange protein) increases by approx. 3-fold the GTP-bound pool of Rab3 isoforms. Rab3-GEP is very similar to MADD, a death domain-containing protein that associates with the type 1 tumour necrosis factor receptor. We observed that the death domain of Rab3-GEP is involved in intramolecular interactions and that deletions or mutations that affect this domain of the protein impair the nucleotide exchange activity towards Rab3. We propose that the death domain of Rab3-GEP acts as a molecular switch and co-ordinates multiple functions of the protein by exchanging its binding partners.