Heterotrimeric guanine nucleotide-regulatory (G) proteins are associated with a variety of intracellular membranes and specific plasma membrane domains. In polarized epithelial LLC-PK1 cells we have shown previously that endogenous G alpha i-2 is localized on the basolateral plasma membrane, whereas G alpha i-3 is localized on Golgi membranes. The targeting of these highly homologous G alpha i proteins to distinct membrane domains was studied by the transfection and expression of chimeric G alpha i proteins in LLC-PK1 cells. Chimeric cDNAs were constructed from the cDNAs for G alpha i-3 and G alpha i-2 and introduced into a pMXX eukaryotic expression vector containing a mouse metallothionein-I promoter. Stably transfected cell lines were produced that expressed either G alpha i-2/3 or G alpha i-3/2 chimeric proteins. Chimeric and endogenous G alpha i proteins were detected in cells using specific carboxy-terminal peptide antibodies. Immunofluorescence staining was used to localize endogenous and chimeric G alpha i proteins in LLC-PK1 cells. The staining of chimeric proteins was detected as an increased intensity of staining on membranes containing endogenous G alpha i proteins. Using confocal microscopy and image analysis we localized G alpha i-2 to a specific sub-domain of the lateral membrane of polarized cells, the chimeric G alpha i-3/2 protein was then shown to colocalize with endogenous G alpha i-2 in the same lateral plasma membrane domain. The chimeric G alpha i-2/3 protein colocalized with endogenous G alpha i-3 on Golgi membranes in LLC-PK1 cells. These results show that chimeric G alpha i proteins were targeted to the same membrane domains as endogenous G alpha i proteins and the specificity of their membrane targeting was conferred by the carboxy-terminal end of the proteins. These data provide the first evidence for specific targeting information contained in the carboxy termini of G alpha i proteins, which appears to be independent of amino-terminal membrane attachment sites in these proteins.

REFERENCES

Adamson
P.
,
Paterson
H. F.
,
Hall
A.
(
1992
).
Intracellular localization of the P21rho proteins.
J. Cell Biol
119
,
617
627
Ali
N.
,
Milligan
G.
,
Evans
W. H.
(
1989
).
Distribution of G-proteins in rat liver plasma-membrane domains and endocytic pathways.
Biochem. J
261
,
905
912
Audigier
Y.
,
Nigam
S. N.
,
Blobel
G.
(
1988
).
Identification of a G protein in rough endoplasmic reticulum of canine pancreas.
J. Biol. Chem
263
,
16352
16357
Ausiello
D. A.
,
Stow
J. L.
,
Cantiello
H. F.
,
Benos
D. J.
(
1992
).
Purified epithelial Na+channel complex contains the pertussis toxin-sensitive Gi-3 protein.
J. Biol. Chem
267
,
4759
4765
Berlot
C. H.
,
Bourne
H. R.
(
1992
).
Identification of effector-activating residues of Gs.
Cell
68
,
911
922
Caplan
M. J.
,
Forbush
B.
,
Palade
G. E.
,
Jamieson
J. D.
(
1990
).
Biosynthesis of the Na,K-ATPase in Madin-Darby canine kidney cells. Activation and cell surface delivery.
J. Biol. Chem
265
,
3528
3534
Chavrier
P.
,
Gorvel
J.-P.
,
Stelzer
E.
,
Simons
K.
,
Gruenberg
J.
,
Zerial
M.
(
1991
).
Hypervariable C-terminal domain of rab proteins acts as a targeting signal.
Nature
353
,
769
772
Crouch
M. F.
(
1991
).
Growth factor-induced cell division is paralleled by translocation of Gito the nucleus.
FASEB J
5
,
200
206
Ercolani
L.
,
Stow
J. L.
,
Boyle
J. F.
,
Holtzman
E. J.
,
Lin
H.
,
Grove
J. R.
,
Ausiello
D. A.
(
1990
).
Membrane localization of the pertussis toxin-sensitive G-protein subunitsi-2 and i-3 and expression of a metallothionein-i-2 fusion gene in LLC-PK1 cells.
Proc. Nat. Acad. Sci. USA
87
,
4637
4639
Gilman
A. G.
(
1987
).
G proteins: transducers of receptor-generated signals.
Annu. Rev. Biochem
56
,
615
649
Gorman
C.
,
Moffat
L.
,
Howard
B.
(
1982
).
Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells.
Mol. Cell. Biol
2
,
1044
1051
Graf
R.
,
Mattera
R.
,
Codina
J.
,
Esyes
M. K.
,
Birnbaumer
L.
(
1992
).
A truncated recombinantsubunit of Gi3 with a reduced affinity for dimers and altered guanosine 5–3-o(thio)triphosphate binding.
J. Biol. Chem
267
,
24307
24314
Hancock
J. F.
,
Cadwallader
K.
,
Paterson
H.
,
Marshall
C. J.
(
1991
).
A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of ras proteins.
EMBO J
10
,
4033
4039
Holtzman
E. J.
,
Soper
B. W.
,
Stow
J. L.
,
Ausiello
D. A.
,
Ercolani
L.
(
1991
).
Regulation of the G-proteini-2 subunit gene in LLC-PK1 renal cells and isolation of porcine genomic clones encoding the gene promoter.
J. Biol. Chem
266
,
1763
1771
Jones
T. L.
,
Simonds
W. F.
,
Merendino
J. J.
Jr.
,
Brann
M. R.
,
Spiegel
A. M.
(
1990
).
Myristoylation of an inhibitory GTP-binding protein alpha subunit is essential for its membrane attachment.
Proc. Nat. Acad. Sci. USA
87
,
568
572
Juhnn
Y. S.
,
Jones
T. L. Z.
,
Spiegel
A. M.
(
1992
).
Amino-and carboxy-terminal deletion mutants of Gsare localized to the particulate fraction of transfected cos cells.
J. Cell Biol
119
,
523
530
Mumby
S. M.
,
Heukeroth
R. O.
,
Gordon
J. I.
,
Gilman
A. G.
(
1990
).
G-protein-subunit expression, myristoylation, and membrane association in COS cells.
Proc. Nat. Acad. Sci.USA
87
,
728
732
Neer
E. J.
,
Pulsifer
L.
,
Wolf
L. G.
(
1988
).
The amino terminus of G proteinsubunits is required for interaction with.
J. Biol. Chem
263
,
8996
9000
Osawa
S.
,
Dhanesekaran
N.
,
Woon
C. W.
,
Johnson
G. L.
(
1990
).
Gi-G s chimeras define the function ofchain domains in control of G protein activation and subunit complex interactions.
Cell
63
,
697
706
Simonds
W. F.
,
Butrynski
J. E.
,
Gautman
N.
,
Unson
C. G.
,
Speigel
A. M.
(
1991
).
G-proteindimer. Membrane targeting requires subunit coexpression and intact C-A-A-X domain.
J. Biol. Chem
266
,
5363
5366
Skorecki
K. L.
,
Verkman
A. S.
,
Ausiello
D. A.
(
1987
).
Cross-talk between stimulatory and inhibitory guanine 5triphosphate binding proteins: Role in activation and desensitization of the adenylate cyclase response to vasopressin.
Biochemistry
26
,
639
647
Sternweiss
P. C.
(
1986
).
The purifiedsubunits of Go and Gi from bovine brain require for association with phospholipid vesicles.
J. Biol. Chem
261
,
631
637
Stevenson
B. R.
,
Siliciano
J. D.
,
Mooseker
M. S.
,
Goodenough
D. A.
(
1986
).
Identification of ZO-1: A high molecular weight polypeptide associated with the tight junction (zonula occludentes) in a variety of epithelia.
J. Cell Biol
103
,
755
766
Stow
J. L.
,
de Almeida
J. B.
,
Narula
N.
,
Holtzman
E. J.
,
Ercolani
L.
,
Ausiello
D. A.
(
1991
).
A heterotrimeric G protein, Gi-3, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PK1 epithelial cells.
J. Cell Biol
114
,
1113
1124
Stow
J. L.
,
Sabolic
I.
,
Brown
D.
(
1991
).
Heterogenous localization of G protein-subunits in rat kidney.
Amer. J. Physiol
261
,
831
–.
Woon
C. W.
,
Sopakar
S.
,
Heasley
L.
,
Johnson
G. L.
(
1989
).
Expression of a Gi/G s chimera that constitutively activates cyclic AMP synthesis.
J. Biol. Chem
63
,
5697
5693
This content is only available via PDF.