RT‐PCR cloning of Rab3 isoforms expressed in peritoneal mast cells

Abstract

Rab proteins are ras-like low molecular mass GTP-binding proteins, which are postulated to act as specific regulators of membrane trafficking m exocytosis and endocytosis. We have previously shown that synthetic peptides, corresponding to the effector domain of Rab3 proteins, stimulate a complete exocytotic response in mast cells. We have used a PCR-clomng strategy to investigate the presence of mRNA encoding Rab3 in mast cells. RNA based PCR was then performed on mast cell RNA using degenerate oligonucleotide primers based on two conserved sequences among Rab3 proteins. However, no PCR products were obtained, even for proteins known to be expressed in high copy numbers in mast cells @-actin and Fc receptor). We have found that the problem resides in the presence of mast cell secretory granule derived heparin, that copurifies with the RNA; heparin has been shown to inhibit the activity of reverse transcriptase and Taq polymerase in PCR. After treating the RNA (obtained from about 500 mast cells) with heparinase, several PCR products of varying size were obtained using primers specific for Rab3 proteins. These products were cloned and sequenced. We have found clones containing sequences that had a 100% homology at the deduced amino acid level to a portion of Rab3B and Rab3D (amino acids 16 to 83).

rab3‐Peptide stimulates exocytosis of the ram sperm acrosome via interaction with cyclic AMP and phospholipase A2 metabolites

“…[16]. Among over 30 rab proteins, rab3 represents a small subfamily (rab3a, rab3b, rab3c, rab3d) expressed in a variety of cells: rab3a mainly in neurons and chromaffin cells; rab3b in pituitary cells, platelets, and mast cells; rab3c in neurons; and rab3d in adipocytes and mast cells [16,19,31]. It has been postulated that rab3 exercises a positive regulatory role in exocytosis because rab3AL, a synthetic peptide corresponding to the rab3 effector domain, stimulates secretion in a variety of cells [20][21][22][23][24].…”

“…However, since the putative effector domains of the different rab3 isoforms are very similar (cf. [31,32]), the response to rab3AL does not allow us to implicate a specific member of the rab3 subfamily (cf. [31]).…”

RasGRP4, a New Mast Cell-restricted Ras Guanine Nucleotide-releasing Protein with Calcium- and Diacylglycerol-binding Motifs

“…7, recombinant RasGRP4 is indeed able to activate H-Ras in vitro. MCs express varied members of the Ras superfamily of GTP-binding proteins (12,26,(42)(43)(44)(45)(46)(47). However, because recombinant hRasGRP3 is able to transfer GTP to GDP-loaded H-Ras, R-Ras, and Rap1 in vitro (18), it remains to be determined which Ras family members RasGRP4 prefers to interact with inside a living MC.…”

Rab3D Is Not Required for Exocrine Exocytosis but for Maintenance of Normally Sized Secretory Granules

“…Originally identified in fat cells (7), Rab3D protein is present in several additional cell types, including secretory cells such as pancreatic (42,55) and parotid (42) acinar cells, mast cells (41), and peptide-secreting cell lines (9). In secretory cells, Rab3D appears to be predominantly localized to secretory granules, thus mirroring the distribution of Rab3A in neurons and neuroendocrine cells.…”