Enhanced bradykinin-stimulated phospholipase C activity in murine embryonic stem cells lacking the G-protein alphaq-subunit.
The gene coding for the G-protein alphaq subunit was interrupted by homologous recombination in murine embryonic stem cells (alphaq-null ES cells) as detected by Southern analysis and reverse-transcriptase PCR. The bradykinin (BK) B2 receptor was stably transfected into wild-type (WT) alphai-2-null and alphaq-null ES cells. The B2 receptor bound BK with high affinity and mobilized Ca2+. BK also activated phospholipase C (PLC), as determined by total inositol phosphate (IP) accumulation in a Bordetella pertussis toxin- and genistein-insensitive manner. In WT and alphai-2-null ES cells, BK increased IP levels approx. 4-fold above baseline. Most interestingly, in alphaq-null ES cells, BK increased IP accumulation approx. 9-fold above baseline. Re-expression of alphaq in alphaq-null ES cells resulted in normalization of the BK-stimulated IP accumulation (4-fold above baseline). These results suggest that the B2 receptor activates PLC through more than one member of the Gq family. Additionally, the absence of alphaq alters the kinetics of IP generation, which may reflect intrinsic characteristics of individual members of the Gq family or a decreased susceptibility to heterologous regulation in the alphaq-null ES cells, thus allowing for a more sustained generation of IP.
Ricupero
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DA
Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, 221 Longwood Ave., Boston, MA 02115, USA.
Polgar
P
P
Taylor
L
L
Sowell
M O
MO
Gao
Y
Y
Bradwin
G
G
Mortensen
R M
RM
D000818:Animals; D015139:Blotting, Southern; D001920:Bradykinin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004622:Embryo, Mammalian / Q000166:cytology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019833:Genistein / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D051379:Mice; D037342:Pertussis Toxin; D016133:Polymerase Chain Reaction; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D014162:Transfection; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007295:Inositol Phosphates; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D019833:Genistein; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D001920:Bradykinin; D002118:Calcium