Expression of a G protein subunit, alpha i-1, in Balb/c 3T3 cells leads to agonist-specific changes in growth regulation.
Cellular receptors for many hormones, neurotransmitters, and growth factors are coupled to intracellular effector enzymes or ion channels through a set of heterotrimeric G proteins. In order to determine whether isoforms of G protein alpha subunits contribute differentially to mitogenic responses, we introduced an alpha subunit isoform, alpha i-1, into Balb/c 3T3 cells that normally lack this subtype. Balb/c 3T3 cells transfected with a plasmid containing cDNA encoding alpha i-1 expressed the alpha i-1 protein as judged both by the appearance of immunoreactive alpha i-1 protein on Western blots and by two-dimensional analysis of the proteins [32P]ADP-ribosylated by pertussis toxin. The amount of alpha i-1 expressed is less than the amount of alpha subunits endogenously present in these cells. Expression of alpha i-1 in the transfected cells slightly blunts stimulation of adenylylcyclase by GTP, guanosine 5'-3-O-(thio)triphosphate, or forskolin, but has no major effect on the ability of thrombin to inhibit the enzyme. In contrast, the expression of alpha i-1 has significant effects on cell growth and on the mitogenic response to thrombin. The alpha i-1-transfected cells have a doubling time that is twice as long as control cells transfected with the same plasmid without a cDNA insert. Despite their slower growth, thymidine incorporation in response to thrombin is greater in transfected than in control cells. Thrombin-stimulated DNA synthesis is sensitive to inhibition by pertussis toxin and is 5-fold more sensitive to inhibition by pertussis toxin in transfected cells than in control cells. The changes are receptor-specific since the mitogenic response to platelet-derived growth factor is indistinguishable between control and transfected cells. These studies suggest that the alpha i subunit composition of the cell may have profound effects on its growth and its response to stimulation through a specific cell surface receptor.
Cui
Z
Z
Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115.
Zubiaur
M
M
Bloch
D B
DB
Michel
T
T
Seidman
J G
JG
Neer
E J
EJ
D016475:3T3 Cells / Q000187:drug effects; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008934:Mitogens; D037342:Pertussis Toxin; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D008934:Mitogens; D010982:Platelet-Derived Growth Factor; D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004247:DNA; D037342:Pertussis Toxin; D013917:Thrombin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases