The stimulatory effect of rabphilin 3a on regulated exocytosis from insulin-secreting cells does not require an association-dissociation cycle with membranes mediated by Rab 3.
Rabphilin 3a is a Rab 3-GTP binding protein concentrated on secretory vesicles of neurons and endocrine cells. There is evidence that rabphilin 3a undergoes cycles of association-dissociation with membranes and that recruitment of rabphilin 3a to secretory vesicles is mediated by Rab 3a, suggesting that rabphilin 3a is a downstream effector of this Rab. In this study we have investigated whether a membrane-anchored form of rabphilin 3a mimics the action of rabphilin 3a on secretion and bypasses the need for Rab 3 function. Overexpression of both wild-type rabphilin 3a and of a transmembrane anchored form of rabphilin 3a stimulated (about 2-fold) evoked secretion of coexpressed human proinsulin from clonal HIT-T15 cells. A similar transmembrane-anchored protein which lacked the Rab 3 binding region stimulated secretion even more effectively. Unexpectedly, a rabphilin 3a deletion mutant missing the Rab 3 binding domain was also stimulatory on secretion, although a further deletion of rabphilin to exclude the first of the two proline-rich regions abolished its stimulatory effect. The first of these two mutants was primarily particulate, while the second mutant was primarily soluble, suggesting that the first proline-rich region of rabphilin 3a plays a role in targeting rabphilin to its site of action. We conclude that the action of rabphilin 3a can be independent of Rab 3 if other mechanisms produce a sufficient concentration of the protein in proximity of exocytotic sites. These results provide new evidence for a fundamental similarity in the mechanisms by which Ras and Rab GTPase produce their distinct physiological effects.
Arribas
M
M
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510/USA.
Regazzi
R
R
Garcia
E
E
Wollheim
C B
CB
De Camilli
P
P
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002135:Calcium-Binding Proteins; D002460:Cell Line; D006224:Cricetinae; D005089:Exocytosis; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D011384:Proinsulin / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D000097624:Rabphilin-3A
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D019204:GTP-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011384:Proinsulin; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D000097624:Rabphilin-3A