Distinct role of Rab3A and Rab3B in secretory activity of rat melanotrophs

1 Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, School of Medicine, and 2 Celica Biomedical Sciences Center, Ljubljana, Slovenia; and 3 Centre National de la Recherche Scientifique (CNRS)-UMR7101, IFR-83, Université P. & M. Curie, and 4 Institut de Biologie-Physico-Chimique, CNRS, UPR 1929, Université Paris 7 Denis Diderot, Paris, France Submitted 9 January 2006 ; accepted in final form 18 June 2006 ABSTRACT Members of the Rab3 (A–D) subfamily of small GTPases are believed to play a key role in regulated exocytosis. These proteins share 80% identity at amino acid level. The question of whether isoforms of Rab3 are functionally redundant was the subject of this study. We used RT-PCR analysis, in situ hybridization histochemistry, and confocal microscope-based analysis of immunocytochemistry to show that rat melanotrophs contain about equal amounts of Rab3A and Rab3B transcripts as well as proteins. Therefore, these cells are a suitable model to study the subcellular distribution and the role of these paralogous isoforms in regulated exocytosis. Secretory activity of single cells was monitored with patch-clamp capacitance measurements, and the cytosol was dialyzed with a high-calcium-containing patch pipette solution. Preinjection of antisense oligodeoxyribonucleotides specific to Rab3A, but not to Rab3B, induced a specific blockage of calcium-dependent secretory responses, indicating an exclusive requirement for Rab3A in melanotroph cell-regulated secretion. Although the injection of purified Rab3B protein was ineffective, the injection of recombinant Rab3A proteins into rat melanotrophs revealed that regulated secretion was stimulated by a GTP-bound Rab3A with an intact COOH terminus and inhibited by Rab3AT36N, impaired in GTP binding. These results indicate that Rab3A, but not Rab3B, enhances secretory output from rat melanotrophs and that their function is not redundant. small GTP-binding proteins; capacitance measurements; immunocytochemistry; exocytosis; reverse transcriptase-polymerase chain reaction Address for reprint requests and other correspondence: R. Zorec, Lab. of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, School of Medicine, PO Box 2211, 1001 Ljubljana, Slovenia (e-mail: robert.zorec{at}mf.uni-lj.si )