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.
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.
Kang
Hatan
H
Life Sciences Division, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, Republic of Korea.
Lee
Won Kyu
WK
Choi
Yun Hui
YH
Vukoti
Krishna Moorthy
KM
Bang
Won Gi
WG
Yu
Yeon Gyu
YG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry*; D006801:Humans; D042541:Intracellular Space / Q000737:chemistry*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry; D011985:Receptors, Serotonin / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D021122:Protein Subunits; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; C097216:serotonin 6 receptor; D019205:GTP-Binding Protein alpha Subunits, Gs