Agonist-induced desensitization and phosphorylation of human 5-HT1A receptor expressed in Sf9 insect cells.
The human 5-HT1A receptor was expressed in Sf9 insect cells to examine desensitization as manifested by agonist-induced uncoupling from G proteins and second messengers. New binding sites were detected after infection of cells with the 5-HT1A receptor-bearing baculovirus. 5-HT1A receptor agonists caused inhibition of cAMP accumulation that could be attenuated by specific receptor antagonists. Brief pretreatment with 5-HT resulted in (1) an uncoupling of receptor from G proteins as evidenced by a loss of high-affinity agonist binding sites and a diminished ability of the receptor to increase incorporation of AA-GTP into endogenous Go alpha-like G proteins, (2) a decreased ability of the receptor to inhibit cAMP accumulation, and (3) increased phosphorylation of the 5-HT1A receptor on serine and threonine residues. Phosphorylation occurred in the presence of a number of cyclic nucleotide dependent kinase inhibitors, and desensitization of the cAMP response occurred in the presence of H-7 and also in cells with prolonged exposure to PMA. Both phosphorylation and desensitization were markedly attenuated by 100 nM and 1 microM heparin and demonstrated similar time courses and concentration-response relationships. Those results demonstrate a close association between agonist-induced desensitization and phosphorylation of the 5-HT1A receptor in Sf9 cells through a pathway that mainly does not involve protein kinase A or C and might involve a G protein-linked receptor kinase.
Nebigil
C G
CG
Department of Medicine (Nephrology Section), Duke University, Durham, North Carolina, USA.
Garnovskaya
M N
MN
Casañas
S J
SJ
Mulheron
J G
JG
Parker
E M
EM
Gettys
T W
TW
Raymond
J R
JR
D017371:8-Hydroxy-2-(di-n-propylamino)tetralin / Q000494:pharmacology; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D017931:DNA Primers / Q000235:genetics; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D011985:Receptors, Serotonin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D044263:Receptors, Serotonin, 5-HT1; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015290:Second Messenger Systems; D012701:Serotonin / Q000494:pharmacology; D018411:Spodoptera
D017931:DNA Primers; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; D011994:Recombinant Proteins; D012701:Serotonin; D015240:Phorbol 12,13-Dibutyrate; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; D000242:Cyclic AMP; D019204:GTP-Binding Proteins