Modulation of 5-MT1A receptor signalling by point-mutation of cysteine351 in the rat Gαo protein

The activity state of G proteins is involved in the ligands maximal responses that can be produced by activating the 5-HT1A receptor (Pauwels et al., 1997). The present study investigated the ligand responses at the recombinant 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 and 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 density 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 binding 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 activity as compared to 5-HT, whereas WAY 100635 acted as a competitive antagonist (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-terminus of the G(alpha o) protein: Ile(351) (93 +/- 3) > Cys(351) (79 +/- 3) > Gly(351) (55 +/- 7). The E-max values (%) of buspirone displayed the following gradient: 69 +/- 5 similar or equal to 62 +/- 8 > 22 +/- 4. For comparison, maximal responses of 8-OH-DPAT and buspirone were enhanced versus 5-HT upon co-expression of the 5-MT1A receptor with the respective G(alpha o) proteins, probably due to an altered receptor: G(alpha o) protein density ratio. 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 produce at these receptors. Moreover, the fusion protein approach allows quantitative comparisons of the intrinsic activities of ligands between one single receptor subtype with different G(alpha) protein subtypes. (C) 1999 Elsevier Science Ltd. All rights reserved.