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.
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.
Ercolani
L
L
Renal Unit, Massachusetts General Hospital, Boston 02129.
Stow
J L
JL
Boyle
J F
JF
Holtzman
E J
EJ
Lin
H
H
Grove
J R
JR
Ausiello
D A
DA
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D005822:Genetic Vectors; D015151:Immunoblotting; D007668:Kidney; D046911:Macromolecular Substances; D008668:Metallothionein / Q000235:genetics*; D010957:Plasmids; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D013552:Swine; D014162:Transfection
D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D012313:RNA; D008668:Metallothionein; D019204:GTP-Binding Proteins