MEMBRANE LOCALIZATION OF THE PERTUSSIS TOXIN-SENSITIVE G-PROTEIN SUBUNIT-ALPHA-I-2 AND SUBUNIT-ALPHA-I-3 AND EXPRESSION OF A METALLOTHIONEIN-ALPHA-I-2 FUSION GENE IN LLC-PK1 CELLS

The renal epithelial cell line LLC-PK1 has topographically distinct regulatory roles for the α subunits of pertussis toxin-sensitive guanine nucleotide regulatory proteins (α(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 α(i) protein family, α(i-2) and α(i-3), which have distinct cellular locations consistent with their diverse functional roles. By using specific α(i) antibodies and immunofluorescence, the α(i-2) subunit was found to be localized to the basolateral membrane, whereas the α(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 α(i-2) gene stably transfected into the LLC-PK1 cells produced an increase in the content of the α(i-2) subunit, which was targeted only to the basolateral membrane. These findings suggest that α(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-α(i-2) fusion gene will provide a model for the study of guanine nucleotide regulatory protein function in epithelia.