Membrane localization of the pertussis toxin-sensitive G-protein subunits alpha i-2 and alpha i-3 and expression of a metallothionein-alpha i-2 fusion gene in LLC-PK1 cells

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 87; no. 12; pp. 4635 - 4639
• Main Authors: Ercolani, L, Stow, J L, Boyle, J F, Holtzman, E J, Lin, H, Grove, J R, Ausiello, D A
• Format: Journal Article
• Language: English
• Published: United States National Acad Sciences 01.06.1990
• Subjects: Animals; Cell Line; Cell Membrane - metabolism; Fluorescent Antibody Technique; Genetic Vectors; GTP-Binding Proteins - analysis; GTP-Binding Proteins - biosynthesis; GTP-Binding Proteins - genetics; Immunoblotting; Kidney; Macromolecular Substances; Metallothionein - genetics; Plasmids; Recombinant Fusion Proteins - biosynthesis; Restriction Mapping; RNA - genetics; RNA - isolation & purification; Swine; Transfection
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.87.12.4635

The renal epithelial cell line LLC-PK1 has topographically distinct regulatory roles for the alpha subunits of pertussis toxin-sensitive guanine nucleotide regulatory proteins (alpha i subunit); these include the inhibition of adenylyl cyclase at the basolateral membrane and the stimulation of Na+ channel activity at the apical membrane. We now report that LLC-PK1 cells contain two members of the alpha i protein family, alpha i-2 and alpha i-3, which have distinct cellular locations consistent with their diverse functional roles. By using specific alpha i antibodies and immunofluorescence, the alpha i-2 subunit was found to be localized to the basolateral membrane, whereas the alpha i-3 subunit was concentrated in the Golgi and was also detectable at low levels on apical membranes in some cells. Induction of a chimeric mouse metallothionein 1-rat or canine alpha i-2 gene stably transfected into the LLC-PK1 cells produced an increase in the content of the alpha i-2 subunit, which was targeted only to the basolateral membrane. These findings suggest that alpha i subunit specificity for effectors may be achieved in polarized renal epithelial cells by their geographic segregation to different cellular membranes. The LLC-PK1 cell stably transfected with the metallothionein-alpha i-2 fusion gene will provide a model for the study of guanine nucleotide regulatory protein function in epithelia.