Carboxyl-methylation of rab3D in the rat pancreatic acinar tumor cell lineAR42J

Rab3D is a small GTPase implicated in regulated exocytosis, and is a marker of secretory granules in exocrine cells. We have previously shown that rab 3D undergoes reversible carboxyl-methylation in adult rat pancreatic acinar cells, and that carboxylmethylation of rab3D is developmentally regulated concomitantly with the maturation of the regulated secretory apparatus in r at pancreas. We also observed that dexamethasone treatment of the rat pancr eatic acinar tumor cell line, AR42J, led to a significant increase in the s ize of the unmethylated pool of a rab3-like protein. The current study was designed to further characterize this rab3-like protein. Here we show that AR42J cells express rab3D, and that the protein focuses on 2D gels as two s pots with pI values of 4.9 and 5.0. Treatment of AR42J cells with N-acetyl- S-geranylgeranyl-L-cysteine, an inhibitor of carboxylmethylation, led to a decrease in the basic form of rab3D and a proportional increase in the acid ic form. In contrast, N-acetyl-S-farnesyl-L-cysteine, which inhibits carbox yl-methylation of farnesylated proteins, had no effect. Lovastatin, an inhi bitor of geranylgeranylation, also induced an accumulation of the acidic fo rm of rab3D. Taken together, these data indicate that rab3D can undergo rev ersible carboxylmethylation in AR42J cells by a geranylgeranyl-specific met hyltransferase. The 2D gel and immunoblotting analyses indicated that dexam ethasone treatment of AR42J cells led to an increase in the proportion of t he unmethylated form of rab3D concurrent to inducing a regulated secretory pathway, similar to the rab3D profile change in developing rat pancreas. Ou r data, along with previous studies done on developing rat pancreas, indica te that the tumor cell line AR42J represents a good model system for studyi ng the regulated secretory pathway, and that carboxyl-methylation of rab3D may play a role in the acquisition of stimulus-secretion coupling. (C) 2001 Academic Press.