The neuropeptide head activator loses its biological acitivity by dimerization.

Bodenmüller, Heinz; Schilling, Elke; Zachmann, B.; Schaller, Heinz

doi:10.1002/j.1460-2075.1986.tb04433.x

Cited by 37 publications

(21 citation statements)

References 19 publications

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“…Monomerisation was achieved by heating a 10 M solution of HA in 0.1 N HCl for 5 minutes to 95°C. After neutralisation with NaOH to pH 7.0, samples were stored frozen at -20°C and used 2-3 times only (Bodenmuller et al, 1986). For labelling Cy3B, 150 nmoles of monomerised HA were lyophilised and dissolved in 100 l dimethylformamide containing 0.2% Nmethylmorpholine.…”

Section: Methodsmentioning

confidence: 99%

The neuropeptide head activator is a high-affinity ligand for the orphan G-protein-coupled receptor GPR37

Rezgaoui

Süsens

Ignatov

et al. 2006

Journal of Cell Science

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The neuropeptide head activator (HA) is a mitogen for mammalian cell lines of neuronal or neuroendocrine origin. HA signalling is mediated by a G-protein-coupled receptor (GPCR). Orphan GPCRs with homology to peptide receptors were screened for HA interaction. Electrophysiological recordings in frog oocytes and in mammalian cell lines as well as Ca2+ mobilisation assays revealed nanomolar affinities of HA to GPR37. HA signal transduction through GPR37 was mediated by an inhibitory G protein and required Ca2+ influx through a channel of the transient receptor potential (TRP) family. It also required activation of Ca2+-dependent calmodulin kinase and phosphoinositide 3-kinase. Respective inhibitors blocked HA signalling and HA-induced mitosis in GPR37-expressing cells. HA treatment resulted in internalisation of GPR37. Overexpression of GPR37 led to aggregate formation, retention of the receptor in the cytoplasm and low survival rates of transfected cells, confirming the notion that misfolded GPR37 contributes to cell death, as observed in Parkinson's disease.

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Section: Methodsmentioning

confidence: 99%

The neuropeptide head activator is a high-affinity ligand for the orphan G-protein-coupled receptor GPR37

Rezgaoui

Süsens

Ignatov

et al. 2006

Journal of Cell Science

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“…Early studies revealed that the purified peptide was active only at very low concentrations, a result that was later supported by analysis of chemically synthesized HA (Birr et al 1981; Schaller and Bodenmuller 1981). Subsequent experiments by molecular sieve chromatography suggested that the peptide forms homodimers with high affinity (K d ≈ 1 nM; Bodenmuller et al 1986), which led to the hypothesis that inactivation at higher concentrations is due to self‐association.…”

mentioning

confidence: 99%

“…Previous attempts at conformational characterization of HA suggested that the molecule contains considerable β‐sheet structure (Bodenmuller et al 1986; Saffrich et al 1989; Fuentes et al 1994). This hypothesis is unusual given that small linear peptides are generally too flexible to adopt well defined conformations in aqueous solution (Wright et al 1988; Creighton 1996).…”

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confidence: 99%

“…This hypothesis is unusual given that small linear peptides are generally too flexible to adopt well defined conformations in aqueous solution (Wright et al 1988; Creighton 1996). Bodenmuller et al (1986) proposed that homodimerization of the peptide occurs via an antiparallel sheet‐like interaction. At the proposed dimer interface, residues Lys 7 through Phe 11 of one HA molecule are postulated to form an extended antiparallel β‐sheet with the same residues of a second HA molecule (Fig.…”

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confidence: 99%

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Reinvestigation of the proposed folding and self‐association of the Neuropeptide Head Activator

Lai

Gellman

2003

Protein Science

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The Neuropeptide Head Activator (HA), pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe (pGlu is pyroglutamic acid), is involved in head-specific growth and differentiation processes in the freshwater coelenterate Hydra attenuata. Peptides of identical sequence have also been isolated from higher-organism tissues such as human and bovine hypothalamus. Early studies by molecular sieve chromatography suggested that HA dimerizes with high affinity (K d ≈ 1 nM). This dimerization was proposed to occur via antiparallel ␤-sheet formation between the Lys 7 -Phe 11 segments in each HA molecule. We conducted biophysical studies on synthetic HA in order to gain insight into its structure and aggregation tendencies. We found by analytical ultracentrifugation that HA is monomeric at low millimolar concentrations. Studies by 1 H-NMR revealed that HA did not adopt any significant secondary structure in solution. We found no NOEs that would support the proposed dimer structure. We probed the propensity of the Lys 7 -Phe 11 fragment to form antiparallel ␤-sheet by designing peptides in which two such fragments are joined by a two-residue linker. These peptides were intended to form stable ␤-hairpin structures with cross-strand interactions that mimic those of the proposed HA dimer interface. We found that the HA-derived fragments may be induced to form intramolecular ␤-sheet, albeit only weakly, when linked by the highly ␤-hairpin-promoting D-Pro-Gly turn, but not when linked by the more flexible Gly-Gly unit. These findings suggest that the postulated mode of HA dimerization and the proposed propensity of the molecule to form discrete aggregates with high affinity are incorrect.

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“…Nolan and colleagues used a dimerization motif (Bodenmuller et al 1986) to constrain the binding site in phage display screens to identify a high-affinity Texas red binding sequence (K d ϭ 25 pM). We next attempted to use this longer hairpin-containing motif to bind to calcium indicators.…”

Section: High-affinity Hairpin Texas Red-binding Motif-containing Pepmentioning

confidence: 99%

Development of new peptide-based tools for studying synaptic ribbon function

Francis

Mehta

Zenisek

2011

Journal of Neurophysiology

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of new peptide-based tools for studying synaptic ribbon function. J Neurophysiol 106: 1028-1037, 2011. First published June 8, 2011 doi:10.1152/jn.00255.2011.-Synaptic ribbons are proteinaceous specialized electron-dense presynaptic structures found in nonspiking sensory cells of the vertebrate nervous system. Understanding the function of these structures is an active area of research (reviewed in Matthews G, Fuchs P. Nat Rev Neurosci 11: 812-822, 2010). Previous work had shown that ribbons could be effectively labeled and visualized using peptides that bind to the synaptic ribbon protein RIBEYE via a PXDLS motif (Zenisek D, Horst NK, Merrifield C, Sterling P, Matthews G. J Neurosci 24: [9752][9753][9754][9755][9756][9757][9758][9759] 2004). Here, we expand on the previous work to develop new tools and strategies for 1) better visualizing synaptic ribbons, and 2) monitoring and manipulating calcium on the synaptic ribbon. Specifically, we developed a new higher-affinity peptide-based label for visualizing ribbons in live cells and two strategies for localizing calcium indicators to the synaptic ribbon. exocytosis; hair cell; retina; synapse THE SYNAPTIC RIBBON IS A PRESYNAPTIC feature found in tonically releasing nonspiking cells of the retina, inner ear, and pineal gland. These proteinaceous structures localize nearby to clusters of voltage-gated calcium channels (Issa and Hudspeth 1996;Midorikawa et al. 2007;Morgans et al. 2005;Zenisek et al. 2004) and tether synaptic vesicles near sites of neurotransmitter release (Matthews and Fuchs 2010). Vesicles tethered to the ribbon itself likely make up most of the vesicles that undergo exocytosis in response to membrane potential changes (LoGiudice et al. 2008;Zenisek 2008). The most abundant protein of the synaptic ribbon is RIBEYE, a splice isoform of the transcriptional corepressor COOH-terminal binding protein 2 (CtBP2) (Schmitz et al. 2000). We previously showed that peptides containing a short sequence, known to bind to the closely homologous CtBP1 (Molloy et al. 1998), can be used as an effective label of synaptic ribbons in living cells when introduced into cells via a patch pipette (Zenisek et al. 2004). This peptide label has proven useful in a number of ribbonsynapse preparations (Bartoletti et al. 2010;Dumitrescu et al. 2009;Frank et al. 2009;LoGiudice et al. 2008;Midorikawa et al. 2007;Schnee et al. 2011). Here, we report new variants of the ribbon-binding peptide that offer the potential to extend the utility of the peptide for experimental use. Specifically, we report 1) two strategies for attaching calcium indicators to the synaptic ribbon, and 2) the synthesis of a ribbon label with higher-affinity and better ribbon detection capabilities. MATERIALS AND METHODS Peptide Synthesis and StorageThe peptides used for covalent conjugation of fluo-4 were synthesized, purified, and purchased from New England Peptide (Ͼ75% purity): X-rhod-binding peptides were synthesized, purified (75% purity), and purchased from GenScript (Piscataway, NJ). New England Pepti...

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The neuropeptide head activator loses its biological acitivity by dimerization.

Cited by 37 publications

References 19 publications

The neuropeptide head activator is a high-affinity ligand for the orphan G-protein-coupled receptor GPR37

The neuropeptide head activator is a high-affinity ligand for the orphan G-protein-coupled receptor GPR37

Reinvestigation of the proposed folding and self‐association of the Neuropeptide Head Activator

Development of new peptide-based tools for studying synaptic ribbon function

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