Molecular analysis of the interaction between the intracellular loops of the human serotonin receptor type 6 (5-HT6) and the α subunit GS protein

The serotonin type 6 (5-HT6) 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-HT6 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-HT6 receptor and the a subunit of G(S) (G alpha(S)). The direct interaction of iL3 and G alpha(S) was demonstrated by co-immunoprecipitation. Furthermore, the kinetic parameters of the interaction between iL3 and G alpha(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 G alpha(S). The critical residues within the iL3 region for the interaction with G alpha(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-HT6 receptor mutants. (c) 2005 Elsevier Inc. All rights reserved.