Pertussis toxin treatment prevents 5-HT(5a) receptor-mediated inhibition of cyclic AMP accumulation in rat C6 glioma cells.
We have investigated the functional coupling of the rat 5HT(5a) receptor subtype to adenylate cyclase in a rat C6 glioma cell line. In 5HT(5a) receptor-transfected cells, 5HT caused a concentration-dependent inhibition of forskolin-stimulated cAMP accumulation, with an EC(50) value of 41 nM and a maximal effect of 57% inhibition. This effect was dependent on the concentration of forskolin used to elevate cAMP levels. Methiothepin (1 mcM), which has high affinity for the 5HT(5a) receptor, antagonized the 5HT(5a) receptor-mediated inhibition, and unmasked a stimulation of cAMP formation similar to that observed in untransfected cells, whereas ketanserin (0.1 mcM) enhanced the inhibitory effect of 5HT. Pertussis toxin treatment (0.5 mcg/ml) completely blocked the inhibitory effect of 5HT on cAMP formation, also revealing increase in cAMP accumulation. Pretreatment of the transfected membranes with pertussis toxin abolished subsequent ADP-ribosylation of a 41 kDa protein, correlating the cAMP effect with a functional uncoupling of an inhibitory G protein from its receptor. These results demonstrate an efficient functional coupling of the rat 5HT(5a) receptor to the inhibition of adenylate cyclase via a pertussis toxin-sensitive G[alpha(i)], inhibitory G-protein.
Thomas
E A
EA
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Matli
J R
JR
Hu
J L
JL
Carson
M J
MJ
Sutcliffe
J G
JG
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D005910:Glioma; D007650:Ketanserin / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000201:enzymology; D037342:Pertussis Toxin; D017434:Protein Structure, Tertiary; D051381:Rats; D011985:Receptors, Serotonin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism; D012702:Serotonin Antagonists / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D037361:Adenylate Cyclase Toxin; D011985:Receptors, Serotonin; D012702:Serotonin Antagonists; D010566:Virulence Factors, Bordetella; C078730:serotonin 5 receptor; D005576:Colforsin; D000246:Adenosine Diphosphate Ribose; D012701:Serotonin; D007650:Ketanserin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000262:Adenylyl Cyclases