High affinity Rab3 binding is dispensable for Rabphilin-dependent potentiation of stimulated secretion.
Rabphilin is a protein that associates with the GTP-bound form of Rab3, a small GTPase that controls a late step in Ca(2+)-triggered exocytosis. Rabphilin is found only in neuroendocrine cells where it co-localises with Rab3A on the secretory vesicle membrane. The Rab3 binding domain (residues 45 to 170), located in the N-terminal part of Rabphilin, includes a cysteine-rich region with two zinc finger motifs that are required for efficient interaction with the small GTPase. To determine whether binding to Rab3A is necessary for the subcellular localisation of Rabphilin, we synthesised point mutants within the Rab3-binding domain. We found that two unique mutations (V61A and L83A) within an amphipathic alpha-helix of this region abolish detectable binding to endogenous Rab3, but only partially impair the targetting of the protein to secretory vesicles in PC12 and pancreatic HIT-T15 cells. Furthermore, both mutants transfected in the HIT-T15 beta cell line stimulate Ca(2+)-regulated exocytosis to the same extent as wild-type Rabphilin. Surprisingly, another Rabphilin mutant, R60A, which possesses a wild-type affinity for Rab3, and targets efficiently to membranes, does not potentiate regulated secretion. High affinity binding to Rab3 is therefore dispensable for the targetting of Rabphilin to secretory vesicles and for the potentiation of Ca(2+)-regulated secretion. The effects of Rabphilin on secretion may be mediated through interaction with another, unknown, factor that recognizes the Rab3 binding domain.
Joberty
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G
Markey Center for Cell Signalling, Health Sciences Center, University of Virginia, Charlottesville, VA 22908, USA. gmj4h@virginia.edu
Stabila
P F
PF
Coppola
T
T
Macara
I G
IG
Regazzi
R
R
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D017354:Point Mutation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020692:rab3 GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D000097624:Rabphilin-3A
D048868:Adaptor Proteins, Signal Transducing; D020558:GTP Phosphohydrolases; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D000097624:Rabphilin-3A