DISTINCT FUNCTIONAL-PROPERTIES OF RAB3A AND RAB3B IN PC12 NEUROENDOCRINE CELLS

Rab3A and Rab3B are highly homologous monomeric GTPases that are putat ive regulators of exocytosis in those tissues in which they are expres sed, We have characterized and directly compared the targeting and fun ctional properties of these isoforms in PC12 neuroendocrine cells. Rab 3A and Rab3B both targeted to norepinephrine (NE)-containing large den se core vesicles (LDCVs) when stably expressed in PC12 cells, as deter mined by immunofluorescence and membrane fractionation. Both Rab3 isof orms also bound to recombinant rabphilin-3A in a GTP-dependent manner. The membrane association of rabphilin-3A was modestly enhanced in Rab 3B-expressing PC12 cells relative to Rab3A-overexpressing cells, In ad dition, overexpression of Rab3A modestly inhibited Ca2+-evoked NE rele ase, whereas Rab3B and a GTP binding mutant (Rab3B N135I) markedly sti mulated the efficiency of [H-3]NE secretion by PC12 cells (i.e. secret ion normalized to total cell radioactivity). Expression of Rab3B and R ab3B N135I increased not only the efficiency of NE secretion but also the accumulation of [H-3]NE into LDCVs (i.e. the secretory cargo avail able for secretion). Neither of these effects was attributable to chan ges in the numbers of LDCVs nor the docking of LDCVs at the plasma mem brane. Our results indicate that Rab3A and Rab3B have similar membrane targeting properties and are capable of interacting with the same put ative downstream effector; i.e. rabphilin-3A. However, these isoforms are functionally distinct monomeric GTPases with Rab3B stimulating a l ate step in Ca2+-evoked secretion when expressed in PC12 cells.