Two active molecular phenotypes of the tachykinin NK1 receptor revealed byG-protein fusions and mutagenesis

The NK1 neurokinin receptor presents two non-ideal binding phenomena, two-c omponent binding curves for all agonists and significant differences betwee n agonist affinity determined by homologous versus heterologous competition binding. Whole cell binding with fusion proteins constructed between eithe r G alpha (s) or G alpha (q), and the NK1 receptor with a truncated tail, w hich secured non-promiscuous G-protein interaction, demonstrated monocompon ent agonist binding closely corresponding to either of the two affinity sta tes found in the wild-type receptor. High affinity binding of both substanc e P and neurokinin A was observed in the tail-truncated G alpha (s) fusion construct, whereas the lower affinity component was displayed by the tail-t runcated G alpha (q) fusion. The elusive difference between the affinity de termined in heterologous versus homologous binding assays for substance P a nd especially for neurokinin A was eliminated in the G-protein fusions, An NK1 receptor mutant with a single substitution at the extracellular end of TM-III(F111S), which totally uncoupled the receptor from G alpha (s) signal ing, showed binding properties that were monocomponent and otherwise very s imilar to those observed in the tail-truncated G alpha (q) fusion construct . Thus, the heterogenous pharmacological phenotype displayed by the NK1 rec eptor is a reflection of the occurrence of two active conformations or mole cular phenotypes representing complexes with the G alpha (s) and G alpha (q ) species, respectively. We propose that these molecular forms do not inter change readily, conceivably because of the occurrence of microdomains or "s ignal-transductosomes" within the cell membrane.