Molecular Analysis of the Interaction Between the Intracellular Loops of the Human Serotonin Receptor Type 6 (5-HT6) and the Alpha Subunit of GS Protein

Biochem Biophys Res Commun. 2005 Apr 8;329(2):684-92. doi: 10.1016/j.bbrc.2005.02.040.

Abstract

The serotonin type 6 (5-HT(6)) receptor is a G-protein coupled receptor (GPCR) coupled to a stimulatory G-protein (G(S)). To identify the structural basis for the interaction of the 5-HT(6) receptor with the G(S) protein, we have dissected the interaction between GST-fusion proteins containing the second intracellular loop (iL2), the third intracellular loop (iL3), or the C-terminal tail of the 5-HT(6) receptor and the alpha subunit of G(S) (Galpha(S)). The direct interaction of iL3 and Galpha(S) was demonstrated by co-immunoprecipitation. Furthermore, the kinetic parameters of the interaction between iL3 and Galpha(S) were measured by surface plasmon resonance, and the apparent dissociation constant was determined to be 0.9 x 10(-6)M. In contrast, the second intracellular loop and C-terminal tail regions showed negligible affinity to Galpha(S). The critical residues within the iL3 region for the interaction with Galpha(S) were identified as conserved positively charged residues near the C-terminus of iL3 by measuring the cellular levels of cAMP produced in response to 5-HT stimulation of cells transfected with 5-HT(6) receptor mutants.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Binding Sites
  • GTP-Binding Protein alpha Subunits, Gs / chemistry*
  • Humans
  • Intracellular Space / chemistry*
  • Molecular Sequence Data
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Tertiary
  • Protein Subunits / chemistry
  • Receptors, Serotonin / chemistry*
  • Recombinant Proteins / chemistry
  • Sequence Homology, Amino Acid
  • Structure-Activity Relationship

Substances

  • Protein Subunits
  • Receptors, Serotonin
  • Recombinant Proteins
  • serotonin 6 receptor
  • GTP-Binding Protein alpha Subunits, Gs