Reconstitution of human 5-hydroxytryptamine5A receptor--G protein coupling in E. coli and Sf9 cell membranes with membranes from Sf9 cells expressing mammalian G proteins.
The human 5-hydroxytryptamine5A (h5-ht5A) receptor was expressed in Escherichia coli (h5-ht5A-E. coli) to verify its pharmacological profile in the absence of G proteins. In addition, the ability of the h5-ht5A receptor to interact with mammalian Gi/o and Gs proteins was investigated by a new reconstitution approach. Agonists displayed lower affinities for h5-ht5A-E. coli than for stably transfected h5-ht5A-HEK 293 cells, due to the absence of G protein coupling in E. coli. Lysergic acid diethylamide behaved as a neutral antagonist, showing equal affinities for the G protein-coupled and the uncoupled receptor. To analyze the G protein coupling behavior of the h5-ht5A receptor, h5-ht5A-E. coli membranes or h5-ht5A-Sf9 insect cell membranes were fused by vortexing to membranes from baculovirus-infected Sf9 cells expressing mammalian G proteins. The ability of the h5-ht5A receptor to differentiate between Gi/Go/Gz and Gs proteins was explored by investigation of agonist binding affinities and agonist-induced stimulation of [35S]GTP gamma S binding. The h5-ht5A receptor failed to interact with Gz and Gs proteins and coupled equally well to Gj and Go proteins to form a complex with high affinity for agonists. Under the applied conditions, however, Gi proteins were found to be better activated than Go proteins in the [35S]GTP gamma S binding assay.
Francken
B J
BJ
Department of Biochemical Pharmacology, Janssen Research Foundation, B-2340 Beerse, Belgium.
Vanhauwe
J F
JF
Josson
K
K
Jurzak
M
M
Luyten
W H
WH
Leysen
J E
JE
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D008322:Mammals; D011485:Protein Binding; D011869:Radioligand Assay; D011985:Receptors, Serotonin / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera; D013462:Sulfur Radioisotopes
D011985:Receptors, Serotonin; D013462:Sulfur Radioisotopes; C078730:serotonin 5 receptor; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins