Two Active Molecular Phenotypes of the Tachykinin NK1 Receptor Revealed by G-protein Fusions and Mutagenesis

• Published in: The Journal of biological chemistry 2001-06, Vol.276 (23), p.19793-19799
• Main Authors: Holst, Birgitte, Hastrup, Hanne, Raffetseder, Ute, Martini, Lene, Schwartz, Thue W.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; COS Cells; GTP-Binding Proteins - genetics; GTP-Binding Proteins - metabolism; Molecular Sequence Data; Mutagenesis; Phenotype; Receptors, Neurokinin-1 - chemistry; Receptors, Neurokinin-1 - genetics; Receptors, Neurokinin-1 - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Signal Transduction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M100621200

The NK1 neurokinin receptor presents two non-ideal binding phenomena, two-component binding curves for all agonists and significant differences between agonist affinity determined by homologous versus heterologous competition binding. Whole cell binding with fusion proteins constructed between either Gαs or Gαq and the NK1 receptor with a truncated tail, which secured non-promiscuous G-protein interaction, demonstrated monocomponent agonist binding closely corresponding to either of the two affinity states found in the wild-type receptor. High affinity binding of both substance P and neurokinin A was observed in the tail-truncated Gαsfusion construct, whereas the lower affinity component was displayed by the tail-truncated Gαq fusion. The elusive difference between the affinity determined in heterologous versushomologous binding assays for substance P and 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 Gαs signaling, showed binding properties that were monocomponent and otherwise very similar to those observed in the tail-truncated Gαq fusion construct. Thus, the heterogenous pharmacological phenotype displayed by the NK1 receptor is a reflection of the occurrence of two active conformations or molecular phenotypes representing complexes with the Gαs and Gαq species, respectively. We propose that these molecular forms do not interchange readily, conceivably because of the occurrence of microdomains or “signal-transductosomes” within the cell membrane.