Human serotonin1B receptor expression in Sf9 cells: phosphorylation, palmitoylation, and adenylyl cyclase inhibition

Biochemistry. 1993 Nov 2;32(43):11727-33. doi: 10.1021/bi00094a032.


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)

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Diphosphate Ribose / metabolism
  • Adenylate Cyclase Toxin
  • Adenylyl Cyclase Inhibitors*
  • Amino Acid Sequence
  • Animals
  • Baculoviridae
  • Binding, Competitive
  • Cells, Cultured
  • Genetic Vectors
  • Molecular Sequence Data
  • Moths
  • Palmitic Acid
  • Palmitic Acids / metabolism
  • Pertussis Toxin
  • Phosphorylation
  • Protein Processing, Post-Translational
  • Protein Structure, Tertiary
  • Receptor, Serotonin, 5-HT1B
  • Receptors, Serotonin / biosynthesis
  • Receptors, Serotonin / drug effects
  • Receptors, Serotonin / metabolism*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / drug effects
  • Recombinant Proteins / metabolism
  • Virulence Factors, Bordetella / pharmacology


  • Adenylate Cyclase Toxin
  • Adenylyl Cyclase Inhibitors
  • Palmitic Acids
  • Receptor, Serotonin, 5-HT1B
  • Receptors, Serotonin
  • Recombinant Proteins
  • Virulence Factors, Bordetella
  • Adenosine Diphosphate Ribose
  • Palmitic Acid
  • Pertussis Toxin