Pharmacological characterisation of the human 5-HT1A receptor and its inhibitory G protein fusions

Fusion proteins between the human 5-HTIA receptor and wild type and pertussis toxin resistant forms of the Gi1alpha and Gi1alpha proteins were constructed and stably expressed in HEK293 cells. These constructs were assessed in terms of second messenger system modulation, receptor-G protein affinity and high affinity GTPase activity and the effects that RGS proteins had on this. In an intact cell assay 5-HT activation of the 5-HT1A receptor-G protein fusions concentration dependently inhibited forskolin stimulated adenylyl cyclase activation. Treatment with pertussis toxin abolished 5-HT1A receptor mediated effects for the receptor alone and the wild type G protein fusions but did not have any effect on cells expressing either the Gly351 or Ile351 Gi1alpha and Gi1alpha 5-HTia receptor-G protein fusion constructs, a result that indicated a lack of coupling to the endogenous pool of G protein by the fusion proteins. Consistent with the results of Bahia et al. (1998) the concentration of 5-HT required to produce a 50% reduction in adenylyl cyclase activity was lowest for the Ile351 Galpha" protein mutant followed by the WT Galpha proteins and was highest for the Gly351 Galpha protein mutants. Although not significant the results, consistent with the literature, also indicated that a lower concentration of 5-HT was required to produce a 50% reduction in adenylyl cyclase activity for the 5-HT1A receptor-Gi1alpha for the Go1alpha fusion proteins. The ability of GDP and suramin to prevent [3H]-8-OH-DPAT specific binding to membranes expressing the 5-HT1A receptor and each of the Gi1alpha and GO1alpha fusion proteins was assessed in radioligand binding assays. The concentrations of either GDP or suramin required to reduce specific binding by 50% were significantly lower for the Ile351 mutants than for the wild type proteins with the Gly351 mutant fusion proteins requiring the highest concentrations. This provided an indication of the affinity of the 5-HT1A receptor for the various G proteins and followed the trend that the more hydrophobic the residue at position 351 of a Gialpha and Goalpha proteins the higher the affinity of the 5-HT1A receptor G protein interaction. The results also indicated that the 5-HTIA receptor had a higher affinity for Gi1alpha than Go1alpha protein interactions. 8-OH-DPAT competition for [3H]-MPPF specific binding to membranes expressing the 5-HT1AGi1alphaC351G/I fusion proteins indicated that a significantly greater proportion of the Ile351 mutant G proteins exist in a 5-HT1A receptor coupled form than the Gly351 G protein mutants. The high affinity GTPase activity of membranes expressing the fusion proteins and the effects of a range of purified RGS protein concentrations was examined. While agonist stimulation of GTPase activity produced an increase in Vmax it did not effect the Km for GTP. The addition of either RGS1 or RGS16 proteins however produced concentration dependent increases in both the Vmax and the Km due to their GAP activity at the Gi1alpha and Go1alpha proteins. The effects of the two RGS proteins were not significantly different, but both proteins produced greater increases in GTPase activity in membranes expressing the Go1alpha fusion protein than membranes expressing the Gi1alpha fusion protein. Neither RGS protein had any significant effect on basal GTPase activity for membranes expressing the Gly351 mutant fusion proteins. Both RGS1 and RGS16 proteins did however produce concentration dependent increases in basal GTPase activity in membranes expressing the Ile351 fusion proteins, a result that reflects the greater intrinsic constitutive GTPase activity of the 5-HT1A receptor-Ile351 G protein fusion constructs. Taking advantage of this latter result, the effects of a range of ligands on high affinity GTPase activity in the absence and presence of a maximal concentration of RGSl protein were examined. In the absence of RGSl protein WAY100635 acted as a weak partial agonist with spiperone, methiothepine, (+)-butaclamol, chlorpromazine and thioridazine acting as inverse agonists. In the presence of RGSl protein, the increase in basal GTPase activity for the Gi1alphaC351l and Go1alphaC351l fusion proteins of 165% and 400% respectively allowed for accurate quantification of ligand effects. Only haloperidol was found to be a neutral ligand at the 5-HT1A receptor-G protein fusions in this assay. Thus, the use of constitutively active receptor-G protein fusions and RGS proteins in a high affinity GTPase assay allowed for accurate discrimination between weak partial, neutral and inverse agonist activity