G protein coupling to the thrombin receptor in Chinese hamster lung fibroblasts.
The specific involvement of G proteins in thrombin receptor-mediated Ca2+ mobilization and DNA synthesis has been studied in single Chinese hamster lung fibroblasts (CCL39 cells) activated by the hexapeptide SFLLRN. Immunoblots performed with antibodies directed against the COOH terminus of G protein alpha subunits revealed that alpha q, alpha i, and alpha o were each present in CCL39 cells. The Ca2+ response to SFLLRN was measured after microinjection of anti-alpha q or anti-alpha o antibodies, which produced a total blockade in 71 and 46% of cells, respectively. A partial inhibition of the SFLLRN-induced Ca2+ response was observed in the remaining cells. The lag time between exposure of the cells to SFLLRN and the onset of Ca2+ mobilization was significantly longer (20-24 s) in cells microinjected with anti-alpha q- or anti-alpha o-antibodies than in control cells microinjected with preimmune serum (9 +/- 1 s). Moreover, the peak height of the Ca2+ response to SFLLRN was decreased by 36 and 73%, respectively in cells microinjected with anti-alpha q or anti-alpha o antibodies. SFLLRN-induced DNA synthesis in growth-arrested CCL39 cells was also inhibited (44-78%) by prior microinjection of anti-alpha q or anti-alpha o antibodies. Anti-alpha 1 antibodies had no effect on the SFLLRN-induced Ca2+ response or on DNA synthesis. These results provide direct evidence that the thrombin receptor in CCL39 cells is coupled to two different types of G proteins, Gq and Go, both causing Ca2+ mobilization and mitogenesis.
Baffy
G
G
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia 19104.
Yang
L
L
Raj
S
S
Manning
D R
DR
Williamson
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000032:analysis / Q000096:biosynthesis; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D019204:GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D008168:Lung / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000494:pharmacology*; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism; D018179:Receptors, Thrombin / Q000187:drug effects / Q000378:metabolism*
D000906:Antibodies; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; C082835:thrombin receptor peptide (42-47); C049925:fura-2-am; D004247:DNA; D019204:GTP-Binding Proteins; D002118:Calcium; D016257:Fura-2