Differential expression of Rab3 isoforms in high- and low-secreting mast cell lines.
The expression of several isoforms of the small-molecular-weight Rab3 GTP-binding proteins is a characteristic feature of all cell types undergoing regulated exocytosis, in which Rab3 proteins are considered to regulate the assembly/disassembly of a fusion complex between granule and plasma membrane in a positive and negative manner through interaction with effector proteins. The pattern of Rab3 protein expression may, therefore, provide a subtle means of regulating exocytosis. To investigate the relationship between Rab3 expression and secretory activity, we assessed the differential expression of individual Rab3 proteins in high- and low-secreting clones of the rat basophilic (RBL) cell line. mRNAs for Rab3 isoforms (a-d) were analyzed by constructing cDNA libraries of high- and low-secreting RBL clones. The relative abundance of mRNAs for Rab3 isoforms was initially determined from the clonal frequency of corresponding cDNA clones. RT-PCR using isoform-specific primers was successfully applied to the quantitation of Rab3a mRNA. The presence of individual Rab3 proteins was revealed by SDS-PAGE and immunoblotting, and also by in situ immunofluorescence confocal microscopy. We present evidence that Rab3a and Rab3c are expressed at high levels in the low-secreting variant, while Rab3d is predominant in the high secretor. Levels of the Rab3 effector proteins, Rabphilin and Noc2, are similar in both RBL cell lines. Subcellular fractionation of unstimulated high and low secretor RBL clones revealed that in both cell types Rab3a has a cytoplasmic location while Rab3d is present in a membrane/organelle fraction containing secretory vesicles. Differences in the pattern of expression of Rab3 isoforms in the two RBL cell lines and their localisation may influence the secretory potential. Furthermore, the presence of Rab3 and effector proteins indicates that the mechanism for regulated exocytosis in cells of mast cells/basophil lineage appears similar to that in pre-synaptic vesicles and pancreatic beta-cells.
Carroll
K
K
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, UK. k.carroll@sheffield.ac.uk
Ray
K
K
Helm
B
B
Carey
E
E
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001491:Basophils / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D015153:Blotting, Western; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000502:physiology; D015723:Gene Library; D005820:Genetic Testing; D047908:Intracellular Signaling Peptides and Proteins; D007536:Isomerism; D008407:Mast Cells / Q000166:cytology / Q000378:metabolism* / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000032:analysis; D010641:Phenotype; D011506:Proteins / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D022142:Secretory Vesicles / Q000737:chemistry; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins / Q000032:analysis; D020692:rab3 GTP-Binding Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D000097624:Rabphilin-3A
D048868:Adaptor Proteins, Signal Transducing; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D012333:RNA, Messenger; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D000097624:Rabphilin-3A; C109307:Rph3al protein, rat