Coupling of the expressed alpha 1B-adrenergic receptor to the phospholipase C pathway in Xenopus oocytes. The role of Go.
alpha 1B-Adrenergic receptor mRNA was injected into Xenopus oocytes, resulting in a norepinephrine-evoked Cl- current. The response was proportional to norepinephrine concentration, blocked by prazosin, and dependent on intracellular Ca2+ derived from inositol trisphosphate-sensitive stores. Oocytes treated with 2 micrograms/ml pertussis toxin showed a time-dependent decrease of the norepinephrine response, taking up to 72 h to show an 80% decrease. Overnight treatment with 10 micrograms/ml pertussis toxin also resulted in 80% reduction. Responses to two other cloned receptors (M1-muscarinic and serotonin-1c) expressed in oocytes were also reduced 50% or more by 72 h of pertussis toxin treatment. Pertussis toxin labeling of the cloned Xenopus alpha o-subunit translated in vitro showed that it was a significantly poorer substrate for pertussis toxin than the two mammalian alpha o-subunits expressed and assayed under identical conditions. This unexpected biochemical behavior of the Xenopus alpha o-subunit is in agreement with the rather unusual treatment conditions required to observe the effects of pertussis toxin on the receptor-evoked Cl- current in the oocyte. Injection of mammalian heterotrimeric G(o) but not Gi3 significantly enhanced the norepinephrine-evoked Cl- current in oocytes. Injection of mixtures of anti-sense oligonucleotides to the Xenopus alpha o-subunit reduced the norepinephrine-evoked Cl- current by 60% within 24 h, compared with oocytes injected with the oligonucleotides encoding sense sequences. These studies indicate that the expressed alpha 1B-adrenergic receptor, like the native muscarinic receptor, utilizes G(o) to couple to the phospholipase C-mediated Cl- current in Xenopus oocytes.
Department of Psychiatry, Mount Sinai School of Medicine, City University of New York, New York 10029.
R D
RD
Blitzer
G
G
Omri
M
M
De Vivo
D J
DJ
Carty
R T
RT
Premont
J
J
Codina
L
L
Birnbaumer
S
S
Cotecchia
M G
MG
Caron
R J
RJ
Lefkowitz
D000818:Animals; D002417:Cattle; D004533:Egtazic Acid / Q000378:metabolism; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007700:Kinetics; D009865:Oocytes / Q000201:enzymology; D037342:Pertussis Toxin; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D014982:Xenopus laevis
D011942:Receptors, Adrenergic, alpha; D010566:Virulence Factors, Bordetella; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins