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

Eur J Cell Biol. 1997 Nov;74(3):209-16.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Amino Acid Sequence
  • Animals
  • CHO Cells
  • Calcium-Binding Proteins*
  • Cell Line
  • Cricetinae
  • Exocytosis*
  • GTP-Binding Proteins / genetics
  • GTP-Binding Proteins / metabolism*
  • Humans
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism
  • Molecular Sequence Data
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Proinsulin / genetics
  • Proinsulin / metabolism*
  • Protein Precursors / genetics
  • Protein Precursors / metabolism
  • Rats
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Subcellular Fractions / metabolism
  • Synaptotagmins
  • Vesicular Transport Proteins
  • rab GTP-Binding Proteins*
  • rab3 GTP-Binding Proteins

Substances

  • Adaptor Proteins, Signal Transducing
  • Calcium-Binding Proteins
  • Membrane Glycoproteins
  • Nerve Tissue Proteins
  • Protein Precursors
  • Recombinant Fusion Proteins
  • Vesicular Transport Proteins
  • rabphilin-3A
  • Synaptotagmins
  • Proinsulin
  • GTP-Binding Proteins
  • rab GTP-Binding Proteins
  • rab3 GTP-Binding Proteins