The human 5-ht5A receptor couples to Gi/Go proteins and inhibits adenylate cyclase in HEK 293 cells.
The G protein coupling of human 5-hydroxytryptamine5A (h5-ht5A) receptors was investigated in stably transfected human embryonic kidney (HEK) 293 cells, using radioligand and guanosine-5'[gamma-35S]thiotriphosphate binding to membranes and cyclic adenosine monophosphate measurements in cells. 5-Carboxamido[3H]tryptamine bound to high- and low-affinity sites on h5-ht5A-HEK 293 cell membranes. Guanylyl-imidodiphosphate addition and pertussis toxin pre-treatment abolished high-affinity binding, indicating coupling to G proteins of the Gi/Go family. [N-methyl-3H]Lysergic acid diethylamide bound to a single site; guanylyl-imidodiphosphate and pertussis toxin did not alter lysergic acid diethylamide affinity. 5-Hydroxytryptamine stimulated guanosine-5'[gamma-35S]thiotriphosphate binding to 130% over basal and this effect was completely abolished by pertussis toxin. Various 5-hydroxytryptamine receptor ligands were tested for inhibition of 5-carboxamido[3H]tryptamine binding and in guanosine-5'[gamma-35S]thiotriphosphate binding assays. 5-Hydroxytryptamine consistently inhibited forskolin-induced cyclic adenosine monophosphate formation by 25% in h5-ht5A-HEK 293 cells; no effect was detected on basal cyclic adenosine monophosphate levels, on intracellular Ca2+ concentration or arachidonic acid release. Our studies demonstrate functional coupling of the h5-ht5A receptor to pertussis toxin-sensitive G proteins and to inhibition of adenylate cyclase activity.
Francken
B J
BJ
Department of Biochemical Pharmacology, Janssen Research Foundation, Beerse, Belgium. bfrancke@janbe.jnj.com
Jurzak
M
M
Vanhauwe
J F
JF
Luyten
W H
WH
Leysen
J E
JE
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D008238:Lysergic Acid Diethylamide / Q000494:pharmacology; D037342:Pertussis Toxin; D011985:Receptors, Serotonin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015290:Second Messenger Systems; D012701:Serotonin / Q000031:analogs & derivatives / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D017366:Serotonin Receptor Agonists; D010566:Virulence Factors, Bordetella; C078730:serotonin 5 receptor; D012701:Serotonin; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008238:Lysergic Acid Diethylamide; C047087:5-carboxamidotryptamine; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins