Subcellular Compartmentalization of Activation and Desensitization of Responses Mediated by NK2 Neurokinin Receptors

Abstract

A functional fluorescent neurokinin NK2 receptor was constructed by joining enhanced green fluorescent protein to the amino-terminal end of the rat NK2 receptor and was expressed in human embryonic kidney cells. On cell suspensions, the binding of fluorescent Bodipy-labeled neurokinin A results in a saturatable and reversible decrease of NK2 receptor fluorescence via fluorescence resonance energy transfer. This can be quantified for nM to M agonist concentrations and monitored in parallel with intracellular calcium responses. On single cells, receptor site occupancy and local agonist concentration can be determined in real time from the decrease in receptor fluorescence. Simultaneous measurement of intracellular calcium responses and agonist binding reveals that partial receptor site occupancy is sufficient to desensitize cellular response to a second agonist application to the same membrane area. Subsequent stimulation of a distal membrane area leads to a second response to agonist, provided that it had not been exposed to agonist during the first application. Together with persistent translocation of fluorescent protein kinase C to the membrane area exposed to agonist, the present data support that not only homologous desensitization but also heterologous desensitization of NK2 receptors is compartmentalized to discrete membrane domains.Neurotransmitters stimulate target cells upon interacting with two major categories of regulatory proteins, ligand-gated ion channels (1-3), and G protein-coupled receptors (4). For ligand-gated ion channels, response activation and termination are mediated by a single molecule that carries the neurotransmitter site and the effector site (1). In contrast, responses mediated by G protein-coupled receptors result from a complex cascade of transient and sequential interactions between the receptor and a series of distinct messengers and effector proteins, which belong to distinct subcellular compartments (5-9).Tachykinins or neurokinins form a family of related neuropeptides found throughout central and peripheral nervous tissues. Their biological activities related to pain transmission (10, 11), smooth muscle contraction, vasodilation, or neurogenic inflammation are mediated by at least three distinct G protein-coupled receptors, NK1, NK2, and NK3, that show preferential binding for the endogenous agonists substance P, neurokinin A (NKA), 1 and neurokinin B (NKB), respectively (12). All three receptor isotypes belong to the family of seven transmembrane regulatory proteins and are coupled to an intracellular calcium release response primarily mediated by the pertussis toxin-insensitive heterotrimeric GTP-binding protein Gq/G11.In the present work, we have used fluorescence resonance energy transfer-based detection of neurokinin A binding to its G protein-coupled receptor, the NK2 tachykinin receptor, to detect real-time interactions on living cells and to study the spatial distribution of response activation and desensitization at the single cell level. Tachykinin NK2 receptors w...

Fluorescence resonance energy transfer to probe human M1 muscarinic receptor structure and drug binding properties

“…Competition assays on EGFP-fused rat NK2 receptors using Bodipy [530/550]-labelled Neurokinin A (Bo-NKA) at 50 nM were performed as previously described (Vollmer et al 1999).…”

“…The quantum yield F D of the donor (0.66 for EGFP and 0.63 for EYFP) was taken from the literature (Tsien 1998). J, the spectral overlap integral for the combined emission of EGFP (or EYFP) and absorbance of Bodipy, was calculated as previously described (Vollmer et al 1999). The efficiency of fluorescence energy transfer (E) was defined as the fractional decrease in EGFP (EYFP) fluorescence due to Bo-PZ binding and was expressed by: Assay miniaturization and drug discovery EGFP S -(D17)hM1-expressing cells, suspended in HEPES-BSA buffer, were distributed (90 000 cells/192 lL/well) using a Biomek 2000 robot (Beckman Coulter, Villepinte, France) into 96-well polystyrene plates (Cliniplates; Thermo Labsystems, Issy-lesMoulineaux, France) previously filled (4 lL/well) with Bo-PZ (80 nM final concentration) and a compound to be tested (at 10 lM).…”

“…Specificity of hM1 and rNK2 receptors fluorescence assays and search for new compounds Bo-PZ binding to EGFP S -(D17)hM1 receptors and Bo-NKA binding to EGFP-rNK2 receptors (Vollmer et al 1999) were tested for their specificity, accuracy, use in drug screening and possible miniaturization. …”

“…None of them (at a micromolar concentration) interfere with Bo-NKA binding to EGFP-rNK2 receptors. Conversely, the more potent tachykinin ligands (SR 48968, Bo-NKA and NKA;Vollmer et al 1999) are inactive or poor inhibitors in the muscarinic FRET assay.…”

“…With the advent of very sensitive fluorescence technologies (Hovius et al 2000), new approaches to study G-protein-coupled receptor (GPCR) structure (Chollet and Turcatti 1999), trafficking (Kallal and Benovic 2000;Gicquiaux et al 2002) and signalling (Milligan 1999), as well as GPCR-protein (Angers et al 2000;Rios et al 2001) and GPCR-ligand (Chollet and Turcatti 1999;Vollmer et al 1999;Ghanouni et al 2001;Palanché et al 2001;Valenzuela-Fernandez et al 2001) interactions, have been developed.…”