EXPRESSION OF RAB3D IN DISPERSED CHIEF CELLS FROM GUINEA-PIG STOMACH

Rab3 proteins are low molecular weight GTP-binding proteins that are e xpressed in neurons and other secretory cells. These proteins are loca lized to secretory vesicles and may play a role in regulated exocytosi s. Presently, four highly homologous Rab3 isoforms (A, B, C, D) have b een identified. We examined the expression of Rab3 isoforms in dispers ed chief cells from guinea pig stomach. Immunoblotting with a specific monoclonal Rab3 antibody detected a 27-kDa protein in chief cell cyto solic and membrane fractions, but staining was more intense in membran e fractions. Using the Rab3 antibody, immunohistochemical staining was detected in chief cells but not in parietal or mucous cells. To deter mine which Rab3 isoform(s) is (are) expressed, chief cell cDNA was obt ained by reverse transcription and subjected to PCR using degenerate p rimers that are specific for rab3 isoforms. The resulting PCR products were cloned and sequenced. The nucleotide sequences obtained were 89% homologous to the nucleotide sequence of mouse rab3D. The deduced ami no acid sequence was identical to that of mouse Rab3D (amino acids 16- 83). Moreover, Rab3D was the only isoform detected in chief cells by t hese methods. To identify rab3 transcripts, the guinea pig rab3D fragm ent obtained by reverse transcription PCR cloning was used as a probe for Northern blotting. The 4.0- and 2.3-kb transcripts identified in c hief cells with the rab3 probe were the same size as those detected by others in mouse adipocytes using a rab3D-specific probe. These result s indicate that Rab3D is expressed in gastric chief cells.