Modulation of 5-HT1A receptor activation by its interaction with wild-typeand mutant G(alpha i3) proteins

Constitutive and agonist-dependent activation of the recombinant human 5-HT 1A receptor(RC: 2.1.5HT.10A) was investigated by co-expression with a rat G (alpha i3) protein in Cos-7 cells. The interaction between the 5-HT1A recep tor and rat G(alpha i3) protein was modulated by substitution of the G(alph a i3), protein site for pertussis toxin-catalysed ADP-ribosylation (cystein e(351)) by each of the natural amino acids. Enhanced basal [S-35]GTP gammaS binding responses (+24 to +189%) were observed with the mutant G(alpha i3) proteins containing at position 351 either a histidine, glutamine, serine, tyrosine or a nonpolar amino acid with the exception of a proline. With ea ch of these mutant G(alpha i3) proteins, spiperone (10 muM), but not WAY 10 0635 (10 muM), reduced (-22 to -60%, p < 0.05) the enhanced basal [S-35]GTP <gamma>S binding response. 5-HT (10 muM)-mediated [S-35]GTP gammaS binding responses attained for some of the mutant G(alpha i3)Cys(351) proteins (Phe , Met, Vat and Ala) more than 300% of that obtained with the wt G(alpha i3) protein. Similar results were also obtained with the prototypical 5-HT1A a gonist 8-OH-DPAT and the partial agonist (-)-pindolol. Fusion proteins asse mbled from the 5-HT1A receptor and either the wt G(alphai)3CYs(351), mutant G(alpha i3)Cys(351)Gly or G(alpha i3)Cys(351)Ile protein displayed similar observations for these ligands as obtained by co-expression of the 5-HT1A receptor with each of these G(alpha i3) proteins. Both the degree of 5-HT1A receptor activation by 8-OH-DPAT and (-)-pindolol, and its inhibition by s piperone, strongly correlate (r(2): 0.78-0.81) with the octanol/water parti tion coefficients of the mutated amino acid at position 351 of the G(alpha i3) protein. The present data also suggest the wt G(alpha i3) protein does not result in maximal activation of the 5-HT1A receptor by the agonists bei ng investigated. (C) 2000 Elsevier Science Ltd. Ail rights reserved.