Human serotonin1B receptor expression in Sf9 cells: phosphorylation, palmitoylation, and adenylyl cyclase inhibition.
Analysis of the primary protein structure of the human serotonin1B (5-HT1B) receptor reveals consensus sites for phosphorylation and a putative site for palmitoylation. To investigate these posttranslational modifications, we have expressed a c-myc epitope-tagged 5-HT1B (m5-HT1B) receptor in Sf9 cells. This strategy enabled receptors to be detected by immunoblot analysis and purified by immunoprecipitation using a monoclonal antibody, 9E10, specific for the c-myc epitope. Agonist radioligand [3H]5-HT binding studies showed that the expressed 5-HT1B and m5-HT1B receptors displayed the characteristic pharmacological profile of the neuronal 5-HT1B receptor. The expressed receptors displayed both high- and low-affinity states for [3H]5-HT, suggesting that the receptors were coupled to endogenous G-proteins. Indeed, agonist binding to the high-affinity receptor state was regulated in the presence of GTP gamma S, Gpp(NH)p, and pertussis toxin. [32P]ADP-ribosylation experiments identified a major approximately 41-kDa ADP-ribosylated protein present in Sf9 membranes that comigrated with partially purified bovine brain Gi alpha/G(o) alpha subunits. Measurements of adenylyl cyclase activity in membranes from cells expressing m5-HT1B receptors showed that serotonergic agonists mediated the inhibition of adenylyl cyclase activity with a rank order of potency comparable to their affinity constants. Immunoblot analysis of membranes prepared from cells expressing m5-HT1B receptors and photoaffinity labeling of the immunoprecipitated material revealed photolabeled species at approximately 95 and at approximately 42 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Pharmacology, University of Toronto, Ontario, Canada.
George
S R
SR
Zastawny
R L
RL
Caron
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M
Bouvier
M
M
Dennis
M
M
O'Dowd
B F
BF
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001667:Binding, Competitive; D002478:Cells, Cultured; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009036:Moths; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D037342:Pertussis Toxin; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D044302:Receptor, Serotonin, 5-HT1B; D011985:Receptors, Serotonin / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D010169:Palmitic Acids; D044302:Receptor, Serotonin, 5-HT1B; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D019308:Palmitic Acid; D037342:Pertussis Toxin