Pancreatic beta-cell protein granuphilin binds Rab3 and Munc-18 and controls exocytosis.
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.
Coppola
Thierry
T
University of Lausanne, Institut de Biologie Cellulaire et de Morphologie, Lausanne, Switzerland 1005.
Frantz
Christian
C
Perret-Menoud
Véronique
V
Gattesco
Sonia
S
Hirling
Harald
H
Regazzi
Romano
R
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D005089:Exocytosis / Q000502:physiology*; D007515:Islets of Langerhans / Q000502:physiology*; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure; D033921:Vesicular Transport Proteins / Q000378:metabolism*; D016335:Zinc Fingers; D020692:rab3 GTP-Binding Proteins / Q000378:metabolism*
D000906:Antibodies; D002352:Carrier Proteins; D017931:DNA Primers; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C512778:Sytl4 protein, rat; D033921:Vesicular Transport Proteins; D020692:rab3 GTP-Binding Proteins