Measurement of the millisecond activation switch of G protein–coupled receptors in living cells

Vilardaga, Jean-Pierre; Bünemann, Moritz; Krasel, Cornelius; Castro, Marián; Lohse, Martin J.

doi:10.1038/nbt838

Cited by 404 publications

(455 citation statements)

References 25 publications

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“…2A and Table 1, which is published as supporting information on the PNAS web site) and approximately 1 order of magnitude lower than that of PTH(1-34) for the WT PTHR (K i ϭ 2.4 Ϯ 0.1 nM) (7). The presence of GFP in the N-terminal domain of the receptor is presumably responsible for the loss in binding affinity.…”

Section: Resultsmentioning

confidence: 94%

“…[Aib 1,3 ,M]PTH (1-14) (peptide sequence Aib-V-Aib-E-I-Q-L-M-H-Q-Har-A-K-W-NH 2 ) was kindly provided by T. Gardella (Massachusetts General Hospital and Harvard Medical School, Boston); [Aib 1,3 ,M] refers to the combined modifications of Aib 1,3 , Gln-10, Har 11 , Ala-12, and Trp-14 on human PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), where Aib is the ␣-amino-isobutyric acid and Har is homoarginine (8).…”

Section: Methodsmentioning

confidence: 99%

“…7). Signals detected by avalanche photodiodes were digitalized with an AD converter (Digidata1322A, Axon Instruments, Union City, CA) and stored on a personal computer by using CLAMPEX 9.0 software (Axon Instruments).…”

Section: Recording Of Ligand-induced Changes In Fretmentioning

confidence: 99%

“…We recently showed that activation of PTHR by binding of PTH 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.…”

mentioning

confidence: 99%

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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.

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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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Section: Resultsmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

Section: Recording Of Ligand-induced Changes In Fretmentioning

confidence: 99%

mentioning

confidence: 99%

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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.

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169

150

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“…Several studies using CFP/YFP FRET and related techniques have investigated GPCR signaling dynamics (55)(56)(57)(58). Studies interrogating heterotrimer activation have used designs wherein activation leads to a reduction in FRET response due to the dissociation of a Gα-CFP and Gβγ-YFP coupled pair, for example, ref.…”

Section: Discussionmentioning

confidence: 99%

Minimal Determinants for Binding Activated Gα from the Structure of a Gα_i1−Peptide Dimer^,

et al. 2006

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G-proteins cycle between an inactive GDP-bound state and active GTP-bound state, serving as molecular switches that coordinate cellular signaling. We recently used phage-display to identify a series of peptides that bind Gα subunits in a nucleotide-dependent manner [Johnston, C. A., Willard, F. S., Jezyk, M. R., Fredericks, Z., Bodor, E. T., Jones, M. B., Blaesius, R., Watts, V. J., Harden, T. K., Sondek, J., Ramer, J. K., and Siderovski, D. P. (2005) Structure 13, 1069-1080]. Here we describe the structural features and functions of KB-1753, a peptide that binds selectively to GDP·AlF 4 − -and GTPγS-bound states of Gα i subunits. KB-1753 blocks interaction of Gα transducin with its effector, cGMP phosphodiesterase, and inhibits transducin-mediated activation of cGMP degradation. Additionally, KB-1753 interferes with RGS protein binding and resultant GAP activity. A fluorescent KB-1753 variant was found to act as a sensor for activated Gα in vitro. The crystal structure of KB-1753 bound to Gα i1 ·GDP·AlF 4 − reveals binding to a conserved hydrophobic groove between switch II and α3 helices, and, along with supporting biochemical data and previous structural analyses, supports the notion that this is the site of effector interactions for Gα i subunits.Heterotrimeric G-proteins serve as critical relays that transmit cues from extracellular stimuli as diverse as neurotransmitters, hormones, photons, and odorants/tastants to intracellular signaling cascades responsible for eliciting specific cellular effects (1,2). In the traditional model of G-protein signaling, cell surface G-protein coupled receptors (GPCRs), upon activation by the aforementioned stimuli, catalyze the exchange of GDP for GTP on the Gα *To whom correspondence should be addressed: UNC Pharmacology, 1106 M.E. Jones Bldg., Chapel Hill, NC 27599-7365. Telephone: 919-843-9363. Fax: 919-966-5640. E-mail: dsiderov@med.unc.edu. Current addresses: Entegrion, 5312 Farrington Rd., Chapel Hill, NC 27517 (J.K.R); Becton Dickinson, 21 Davis Dr., RTP, NC 27709 (R.B.); Amgen Inc., 1201 Amgen Court W., Seattle, WA 98119 (Z.F.). † This work was supported by NIH R01 GM074268 (D.P.S.) and EY12859 (V.Y.A.). C.A.J. was supported by an NIH postdoctoral fellowship (1 F32 GM076944). V.Y.A. is the recipient of the Senior Scientific Investigator Award from Research to Prevent Blindness Inc. Coordinates of the KB-1753/Gα i1 ·GDP·AlF 4 − complex were deposited in the Protein Data Bank (accession code 2G83). 1 Abbreviations: AlF 4 − , aluminum tetrafluoride; CFP, cyan fluorescent protein; cGMP, cyclic guanosine monophosphate; FRET, fluorescence resonance energy transfer; GAP, GTPase-accelerating protein; GDP, guanosine diphosphate; GEF, guanine nucleotide exchange factor; GMP, guanosine monophosphate; GPCR, G protein-coupled receptor; GTP, guanosine triphosphate; PDE, phosphodiesterase; RGS, regulator of G-protein signaling; ROS, rod outer segment; SPR, surface plasmon resonance; YFP, yellow fluorescent protein. subunit. This results in adoption of the active, GTP-bou...

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Energy transfer–based approaches to study G protein–coupled receptor dimerization and activation

Jockers

Bouvier

Marullo

2005

Encyclopedia of Genetics, Genomics, Proteomics and Bioinformatics

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Measurement of the millisecond activation switch of G protein–coupled receptors in living cells

Cited by 404 publications

References 25 publications

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

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

Minimal Determinants for Binding Activated Gα from the Structure of a Gα_i1−Peptide Dimer^,

Energy transfer–based approaches to study G protein–coupled receptor dimerization and activation

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Measurement of the millisecond activation switch of G protein–coupled receptors in living cells

Cited by 404 publications

References 25 publications

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

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

Minimal Determinants for Binding Activated Gα from the Structure of a Gαi1−Peptide Dimer,

Energy transfer–based approaches to study G protein–coupled receptor dimerization and activation

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Minimal Determinants for Binding Activated Gα from the Structure of a Gα_i1−Peptide Dimer^,