The Neurokinin A Receptor Activates Calcium and cAMP Responses through Distinct Conformational States

Palanché, Tania; Ilien, Brigitte; Zoffmann, Sannah; Reck, Marie-Pierre; Bucher, Bernard; Edelstein, Stuart J.; Galzi, Jean‐Luc

doi:10.1074/jbc.m104363200

Cited by 121 publications

(128 citation statements)

References 45 publications

(55 reference statements)

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“…They suggest that this active conformation does not involve the same movement of TM6 around Pro-288 6.50 that occurs upon activation of the ␤ 2 AR by catecholamines. These studies provide mechanistic insight into previous fluorescence lifetime experiments demonstrating that salbutamol and isoproterenol induce distinct conformational changes in the cytoplasmic end of TM6 of the ␤ 2 AR (3,9,27).…”

Section: Discussionmentioning

confidence: 88%

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Probing the β2 Adrenoceptor Binding Site with Catechol Reveals Differences in Binding and Activation by Agonists and Partial Agonists

Swaminath

Deupí²,

Lee

et al. 2005

Journal of Biological Chemistry

246

212

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The ␤ 2 adrenergic receptor (␤ 2 AR) is a prototypical family A G protein-coupled receptor (GPCR) and an excellent model system for studying the mechanism of GPCR activation. The ␤ 2 AR agonist binding site is well characterized, and there is a wealth of structurally related ligands with functionally diverse properties. In the present study, we use catechol (1,2-benzenediol, a structural component of catecholamine agonists) as a molecular probe to identify mechanistic differences between ␤ 2 AR activation by catecholamine agonists, such as isoproterenol, and by the structurally related non-catechol partial agonist salbutamol. Using biophysical and pharmacologic approaches, we show that the aromatic ring of salbutamol binds to a different site on the ␤ 2 AR than the aromatic ring of catecholamines. This difference is important in receptor activation as it has been hypothesized that the aromatic ring of catecholamines plays a role in triggering receptor activation through interactions with a conserved cluster of aromatic residues in the sixth transmembrane segment by a rotamer toggle switch mechanism. Our experiments indicate that the aromatic ring of salbutamol does not activate this mechanism either directly or indirectly. Moreover, the non-catechol ring of partial agonists does not interact optimally with serine residues in the fifth transmembrane helix that have been shown to play an important role in activation by catecholamines. These results demonstrate unexpected differences in binding and activation by structurally similar agonists and partial agonists. Moreover, they provide evidence that activation of a GPCR is a multistep process that can be dissected into its component parts using agonist fragments.

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Section: Discussionmentioning

confidence: 88%

“…Using Ligand Fragments to Dissect the Process of Ligand Binding and Activation-Evidence from several studies suggests that agonists activate GPCRs through a series of conformational intermediates (3,9,(27)(28)(29)(30). These studies form the basis for our current hypothesis regarding the mechanism of activation (9).…”

Section: Discussionmentioning

confidence: 99%

Probing the β2 Adrenoceptor Binding Site with Catechol Reveals Differences in Binding and Activation by Agonists and Partial Agonists

Swaminath

Deupí²,

Lee

et al. 2005

Journal of Biological Chemistry

246

212

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“…The slope of k obs fast versus the concentration of hormone gave an association rate constant k 1 ϭ (0.7 Ϯ 0.1) ϫ 10 6 M Ϫ1 ⅐s Ϫ1 , which is too low to be under diffusion control (10 8 to 10 9 M Ϫ1 ⅐s Ϫ1 ) but was within the range for interactions between protein-protein or peptide-receptor (10 5 to 10 6 M Ϫ1 ⅐s Ϫ1 ) (15,16). The intercept with the ordinate axis gave a dissociation rate constant of k Ϫ1 ϭ 0.04 Ϯ 0.01 s Ϫ1 , reflecting the slow dissociation of the bound hormone.…”

Section: Two-mentioning

confidence: 99%

Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism

Castro

Nikolaev²,

Palm³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

168

150

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FRET ͉ G protein-coupled receptor ͉ time-resolved signaling ͉ real-time binding ͉ ligand-receptor interaction P arathyroid hormone (PTH) is an 84-aa peptide that regulates concentrations of calcium and phosphate in blood and is of paramount importance in renal and bone metabolism (1). PTH is secreted from the parathyroid glands in response to hypocalcemia and elicits its physiological effects in kidney and bone through the activation of a G protein-coupled receptor, the PTH͞PTH-related receptor (PTHR) (2). In response to PTH this receptor leads to activation of adenylyl cyclases (via G s protein) that in turn increase the intracellular second messenger cAMP, which activates the protein kinase A (PKA)-signaling pathway. PTHR also activates the phospholipase C͞protein kinase C-signaling pathways (via G q protein), although less efficiently than the PKA-signaling pathway (3). PTH(1-34), a synthetic PTH fragment with the first 34 aa of PTH that holds the same pharmacological properties in regard to the regulation of calcium homeostasis as the full-length hormone (4), has recently become a drug (the generic name is teriparatide) approved by the U.S. Food and Drug Administration to treat osteoporosis (5, 6). The resolution of the mode of interaction of PTH(1-34) to its receptor is critical for understanding the mechanism of action of the hormone and for further progressing in drug development to treat osteoporosis.We recently showed that activation of PTHR by binding of PTH(1-34) proceeds by fast conformational changes (time constant Ϸ 1 s) as the receptor switches from a resting to an active state (7). To further our understanding on the PTHR interaction process we resolved the temporal events of PTH(1-34) binding to PTHR in living cells. We measured kinetics of association by FRET between tetramethyl-rhodamine (TMR) (the acceptor fluorophore) conjugated to PTH(1-34) and GFP (the donor fluorophore) inserted into the N-terminal domain of the receptor (Fig. 1A). This study reports the analysis of these kinetics and reveals a two-step process for PTH(1-34) binding, where the first phase mirrored agonist binding to the receptor N-domain with a fast association rate and the second phase reflected association to the J-domain, exhibiting much slower kinetics but temporally coupled to receptor activation. Materials and MethodsPeptides. Human PTH(1-34) was purchased from Bachem, and the radioligand [ 125

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“…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.…”

mentioning

confidence: 99%

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

Ilien

Franchet

Bernard

et al. 2003

Journal of Neurochemistry

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Human M1 muscarinic receptor chimeras were designed (i) to allow detection of their interaction with the fluorescent antagonist pirenzepine labelled with Bodipy [558/568], through fluorescence resonance energy transfer, (ii) to investigate the structure of the N-terminal extracellular moiety of the receptor and (iii) to set up a fluorescence-based assay to identify new muscarinic ligands. Enhanced green (or yellow) fluorescent protein (EGFP or EYFP) was fused, through a linker, to a receptor N-terminus of variable length so that the GFP barrel was separated from the receptor first transmembrane domain by six to 33 amino-acids. Five fluorescent constructs exhibit high expression levels as well as pharmacological and functional properties superimposable on those of the native receptor. Bodipy-pirenzepine binds to the chimeras with similar kinetics and affinities, indicating a similar mode of interaction of the ligand with all of them. From the variation in energy transfer efficiencies determined for four different receptor-ligand complexes, relative donor (EGFP)-acceptor (Bodipy) distances were estimated. They suggest a compact architecture for the muscarinic M1 receptor amino-terminal domain which may fold in a manner similar to that of rhodopsin. Finally, this fluorescence-based assay, prone to miniaturization, allows reliable detection of unlabelled competitors.

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The Neurokinin A Receptor Activates Calcium and cAMP Responses through Distinct Conformational States

Cited by 121 publications

References 45 publications

Probing the β2 Adrenoceptor Binding Site with Catechol Reveals Differences in Binding and Activation by Agonists and Partial Agonists

Probing the β2 Adrenoceptor Binding Site with Catechol Reveals Differences in Binding and Activation by Agonists and Partial Agonists

Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism

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

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