Modulation of 5-MT1A receptor signalling by point-mutation of cysteine(351) in the rat G(alpha o) protein

The activity state of G proteins is involved in the ligands maximal respons es that can be produced by activating the 5-HT1A receptor (Pauwels et al., 1997). The present study investigated the ligand responses at the recombina nt h 5-HT1A receptor (RC: .1.5HT.01A) as mediated by the G(alpha o) protein . Therefore, a fusion protein was constructed between the 5-HT1A receptor a nd a pertussis toxin resistant rat G(alpha o)Cys(351)Gly mutant protein to define its pharmacological properties at a receptor: G(alpha o) protein den sity ratio of 1. Pertussis toxin treatment (100 ng/ml) affected neither the expression of the 5-HT1A receptor fusion protein as measured by [H-3] MPPF (3.0 +/- 0.7 pmol/mg protein) nor the 5-MT-mediated [S-35]GTP gamma S bind ing response (146 +/- 34 fmol/mg protein) in Cos-7 cells. 8-OH-DPAT (E-max: 55 +/- 7%) and buspirone (E-max: 22 +/- 4%) yielded partial agonist activi ty as compared to 5-HT, whereas WAY 100635 acted as a competitive antagonis t (pK(B): 9.75 +/- 0.17). The magnitude of the 8-OH-DPAT response (E-max, % ) was highly dependent on the nature of the amino acid 351 in the C-terminu s of the G(alpha o) protein: Ile(351) (93 +/- 3) > Cys(351) (79 +/- 3) > Gl y(351) (55 +/- 7). The E-max values (%) of buspirone displayed the followin g gradient: 69 +/- 5 similar or equal to 62 +/- 8 > 22 +/- 4. For compariso n, maximal responses of 8-OH-DPAT and buspirone were enhanced versus 5-HT u pon co-expression of the 5-MT1A receptor with the respective G(alpha o) pro teins, probably due to an altered receptor: G(alpha o) protein density rati o. In conclusion, residue 351 of the rat G(alpha o) protein is involved in determining the magnitude of 5-MT1A receptor activation that ligands can pr oduce at these receptors. Moreover, the fusion protein approach allows quan titative comparisons of the intrinsic activities of ligands between one sin gle receptor subtype with different G(alpha) protein subtypes. (C) 1999 Els evier Science Ltd. All rights reserved.