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

Abstract

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 G␣ s or G␣ q and the NK1 receptor with a truncated tail, which secured nonpromiscuous 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 G␣ s fusion construct, whereas the lower affinity component was displayed by the tail-truncated G␣ q fusion. The elusive difference between the affinity determined in heterologous versus homologous 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 G␣ s signaling, showed binding properties that were monocomponent and otherwise very similar to those observed in the tail-truncated G␣ q 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 G␣ s and G␣ q 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.Many G-protein-coupled 7TM 1 (transmembrane segment) receptors have the capacity to interact with multiple G-proteins and thus regulate more than one effector pathway. Some receptors preferentially couple to one G-protein, but in the presence of higher, non-physiological concentrations of agonist or in, for example reconstitution assays, these receptors are able to couple to other G-proteins (1, 2). Other more promiscuous receptors couple functionally to more than one G-protein in the physiological range of agonist concentration (3-6). For example, the neurokinin NK1 receptor is coupled to two different signaling pathways: a G␣ q pathway, which activates phospholipase C ␤, thereby initiating inositol phosphate formation and a G␣ s pathway, which induces cAMP formation. Both effector systems are activated by nanomolar physiological concentrations of substance P (6). Other endogenous neurokinin peptides such as NKA and NKB as well as artificial peptide ligands such as septide can act as high affinity agonists on the NK1 receptor (7). Interestingly, in whole cell binding experiments, the NK1 receptor displays certain characteristics that indicate that the receptor in the cell membrane occurs in more than one high affinity active molecular form. First, competition binding experiments using the agonist substance P as a tracer reveal two-component binding curves for all agonists, indicating the occurrence of ...

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