Mutations in the Extracellular Amino-terminal Domain of the NK2 Neurokinin Receptor Abolish cAMP Signaling but Preserve Intracellular Calcium Responses

Abstract

G-protein-coupled receptors (GPCRs)1 represent the largest family of cell surface receptors and have multiple effects on the activated cell. Despite the variety of molecular types of ligands (hormones, proteins, small peptides, ions, lipids, and sensory stimuli such as odorants, pheromones, and photons), GPCRs share homology in their core domain composed of seven transmembrane ␣-helices. The conservation of the same overall hydrophobic structure exemplifies the essential role of this structure for the activation process.GPCR activation promotes GDP to GTP exchange on the ␣ subunit of the heterotrimeric G protein associated with its cytoplasmic loops, thereby initializing the intracellular cascade of signaling events. Among the theoretical models describing the activation mechanism, the two-state allosteric model of activation has been proposed, following the discovery of inverse agonists and of constitutive activity for GPCRs (1-4). In this model, a GPCR preexists in equilibrium between an active and an inactive state in the absence of ligand. As such, agonists, higher affinity for the active state having displace the equilibrium toward activation, whereas inverse agonists have higher affinities for the inactive state. Constitutive activity is explained by an intrinsic tendency for a receptor to spontaneously isomerize toward the active conformation. Although the multiple conformational states theory is an attractive model for describing GPCR activation, there is a need for more data correlating the parameters of possible intermediate conversion states with physiological responses.Indeed, several GPCRs have been reported to mediate multiple signaling pathways through activation of different heterotrimeric G proteins: the receptors for dopamine D1 (G s and G o (5), G s and G q (6)) and D5 (G s and G z (7)), the receptors for the parathyroid hormone (G s , G q , and G i (8)), for the corticotropinreleasing hormone (G s , G i , G q , G o , and G z (9)), for the melaninconcentrating hormone (G i and G o and maybe G q (10)), for the vasoactive intestinal peptide (G s and G i (11)), for prostacyclin (G s , G i , and G q (12)), the adenosine A1 receptor (G o and G i (13)), the ␤ 2 -adrenergic receptor (G s and G i (14)), the muscarinic m3 receptor (G q and G 12 (15)), the 5-hydroxytryptamine receptor type 4; Bpa, G o , and G s (16)), the endothelin subtype B receptor (G i , G o , and G q (17, 18)). These different couplings suggest that GPCRs may exist in more than a single active state.We have previously addressed the question of multiple receptor states by using fluorescence resonance energy transfer (FRET) to monitor ligand binding in parallel with cellular responses. A chimeric green fluorescent neurokinin NK2 receptor, EGFP-NK2R was expressed in HEK293 cells. Two of its neuropeptide agonists, the decapeptide neurokinin A (NKA) and its truncated form, NKA-(4 -10), were covalently linked to the fluorophore Texas Red (TR) (19). Kinetic measurements of the associations of TR1-NKA and TR7-NKA-(4 -10) to the EGFP-NK2 re...

Post-activation-mediated Changes in Opioid Receptors Detected by N-terminal Antibodies

“…The majority of studies examining activity induced conformational changes in GPCRs have focused, thus far, on transmembrane helices or intracellular regions (17,18). Relatively few studies have examined the involvement of the extracellular region in general and the N-terminal region in particular in this process (19). Studies with receptors with a long N-terminal tail such as family C receptors (4,5) have led to the proposal that the N terminus is organized into a Venus flytrap module that undergoes specific ligand-induced conformational changes (4,5).…”

Dynamic Confinement of NK2 Receptors in the Plasma Membrane

“…Seven fractions were collected from each gradient with fraction 1 having the lightest density. To analyze the content in fluorescently modified fusion proteins (either EGFP-NK2 receptors or EYFP-GPI or EYFP-CD46), each fraction of the density gradient was submitted to immunoprecipitation with polyclonal anti-GFP antibodies and the immunoprecipitated material was separated on polyacrylamide gels, blotted, and revealed with monoclonal anti-GFP antibodies (8). To analyze the content of each fraction of the density gradient in endogenous caveolin-1, each supernatant of the anti-GFP immunoprecipitation was trichloroacetic acid-precipitated, separated on polyacrylamide gels, blotted, and revealed with polyclonal anti-caveolin-1 (Santa Cruz Biotechnology) (22).…”

Les récepteurs couplés aux protéines G

“…Il n'en est pas moins vrai qu'un modèle à deux états ne peut pas rendre compte de la complexité des voies de signalisation des RCPG. De nombreuses études fonctionnelles suggèrent en effet l'existence d'états actifs multiples, stabilisés de manière différentielle par les ligands orthostériques (ou allostériques) et discriminés par des partenaires de signalisation intracellulaires différents [3,8,9]. Les données structurales obtenues à partir des RCPG cristallisés dans des états actif et inactif [10,32] (➜) complé-tées par des études biophysiques Ainsi ont émergé : (1) l'idée de rechercher des composés ciblant des domaines de RCPG moins conservés, (2) la nécessité de comprendre les bases moléculaires sous-jacentes à la sélection d'états conformationnels et fonctionnels spécifiques pour chaque RCPG, et (3) la possibilité d'utiliser ces outils et ces connaissances pour atteindre un niveau supérieur de sélectivité et de régulation de l'activité physiologique de ces récepteurs.…”

“…Néanmoins, et à de rares exceptions près, tous ces ligands allostériques ralentissent (à des degrés divers) les cinétiques à la fois On observe ensuite une phase de liaison plus lente, qui correspond à l'isomérisation des complexes C 1 L en complexes C 2 L de plus haute affinité. La coïncidence temporelle entre les deux étapes de liaison et la production des signaux intracellulaires (Ca 2+ et AMPc) a permis de discerner les états conformationnels et de les associer à des états fonctionnels [8,16]. AC : adénylate cyclase ; PLC : phospholipase C. B. Modulation allostérique de l'activation du récepteur NK2 des tachykinines.…”