Peptide synthesis. Part 1. Preparation and use of polar supports based on poly(dimethylacrylamide)

Arshady, Reza; Atherton, Eric; Clive, Derek L. J.; Sheppard, R. C.

doi:10.1039/p19810000529

Cited by 156 publications

(86 citation statements)

References 3 publications

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“…Peptides 6, 7, and 8 were acetylated on the N terminus and were synthesized as C-terminal amides. Polydimethylacrylamide-based PL-DMA resin (Varian, Inc., Palo Alto, CA) was treated overnight with neat ethylenediamine as described by Arshady et al (1981). After thoroughly washing the resin with DMF/CH 2 Cl 2 , Fmoc-Rink amide linker was added in 3-fold excess, as calculated from the nominal loading of 1 mmol/g.…”

Section: Methodsmentioning

confidence: 99%

Role for the Regulator of G-Protein Signaling Homology Domain of G Protein-Coupled Receptor Kinases 5 and 6 in β2-Adrenergic Receptor and Rhodopsin Phosphorylation

Morgan¹,

Yao²,

Tran³

et al. 2009

Mol Pharmacol

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Phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) is a major mechanism of desensitization of these receptors. GPCR activation of GRKs involves an allosteric site on GRKs distinct from the catalytic site. Although recent studies have suggested an important role of the N-and C-termini and domains surrounding the kinase active site in allosteric activation, the nature of that site and the relative roles of the RH domain in particular remain unknown. Based on evolutionary trace analysis of both the RH and kinase domains of the GRK family, we identified an important cluster encompassing helices 3, 9, and 10 in the RH domain in addition to sites in the kinase domain. To define its function, a panel of GRK5 and -6 mutants was generated and screened by intactcell assay of constitutive GRK phosphorylation of the ␤ 2 -adrenergic receptor (␤2AR), in vitro GRK phosphorylation of lightactivated rhodopsin, and basal catalytic activity measured by tubulin phosphorylation and autophosphorylation. A number of double mutations within helices 3, 9, and 10 reduced phosphorylation of the ␤2AR and rhodopsin by 50 to 90% relative to wild-type GRK, as well as autophosphorylation and tubulin phosphorylation. Based on these results, helix 9 peptide mimetics were designed, and several were found to inhibit rhodopsin phosphorylation by GRK5 with an IC 50 of ϳ30 M. In summary, our studies have uncovered previously unrecognized functionally important sites in the regulator of G-protein signaling homology domain of GRK5 and -6 and identified a peptide inhibitor with potential for specific blockade of GRK-mediated phosphorylation of receptors.To understand the mechanism underlying the activation of GRKs by the GPCRs, it is essential to identify functional sites in the GRKs involved in their activation. The serine/ threonine GRK family includes seven members, GRK1-7, classified into three subfamilies on the basis of their sequence homology: the rhodopsin kinase subfamily (GRK1 and -7), activities of which are restricted to the visual system; the ␤-adrenergic receptor kinase subfamily (GRK2 and -3); and the GRK4 subfamily (GRK4 -6) (Krupnick and Benovic, 1998;Pitcher et al., 1998). Crystal structures have been determined for GRK2 in complex with the ␤␥ and G␣ q subunits of G proteins (Lodowski et al., 2003(Lodowski et al., , 2005(Lodowski et al., , 2006Tesmer et al., 2005), for GRK6 bound to 5Ј-adenylylimidodiphosphate (Lodowski et al., 2006), and recently for six crystal structures of rhodopsin kinase (Singh et al., 2008). These structures all seem to be in the inactive state. Thus, neither the activestate conformation of GRKs nor how they interface with GPCRs is known.Membrane localization and activation of GRKs are complex, involving several domains within the kinase. It was shown that the GRK2 N-terminal fragment (residues 45-178) coimmunoprecipitated with metabotropic glutamate receptor 1 (Dhami et al., 2002) and that a single mutation in

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Section: Methodsmentioning

confidence: 99%

Role for the Regulator of G-Protein Signaling Homology Domain of G Protein-Coupled Receptor Kinases 5 and 6 in β2-Adrenergic Receptor and Rhodopsin Phosphorylation

Morgan¹,

Yao²,

Tran³

et al. 2009

Mol Pharmacol

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“…Polyacrylamide resin [4]. (0.3 mmol sarcosine/g resin) was from Imperial Chemical Industries Ltd. Fmoc-Gln-p-nitrophenylester and Fmoc-Leu were purchased from Chemical Dynamics Co. For the preparation of Fmoc-MeGly and Fmoc-MePhe see [l] and for Fmoc-Phe (I) see [5].…”

Section: Methodsmentioning

confidence: 99%

Synthesis and metabolic stability of a tritium‐labelled substance P analogue

Sandberg¹,

Hanley²,

Watson³

et al. 1982

FEBS Letters

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“…[20][21][22][23] We will discuss the synthesis of the aminofunctional SLURPS first, followed by linker conjugation, as this has not been reported before, followed by our SPPS of Leu-enkephalin.…”

Section: Introductionmentioning

confidence: 99%

Novel Non-PEG Derived Polyethers as Solid Supports. 2. Solid-Phase Synthesis Studies

et al. 2007

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Novel non-PEG derived polyether resins, coined SLURPS (Superior Liquid Uptake Resins forPolymer-supported Synthesis), were studied for their performance in solid-phase synthesis. Novel amino functional resins, SLURPS-NH 2 , were prepared with a loading of up to 8.5 mmol/g and employed successfully in the solid-phase synthesis of Leu-Enkephalin. The peptide was obtained with the same 2 purity when compared to its synthesis with commercial standard poly(dimethyl acrylamide) resins.Furthermore we show loading and cleavage of aromatic carboxylic acids in excellent yield. The advantageous solvent compatibility of our support was demonstrated through the biphasic dihydroxylation of alkenes with OsO 4 in t-BuOH/water mixtures producing bound 1,2-diols and synthesis and removal of a bound oxime using ethanol/water mixtures both in excellent yields. Reactions were easily monitored by gel-phase NMR and FTIR These results show that SLURPS are very well suited for organic transformations using highly polar solvent mixtures and reagents and this at much higher loading levels than standard amphiphilic resins of similar solvent compatibility. Graphical Abstract:O OH SLURPS

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Peptide synthesis. Part 1. Preparation and use of polar supports based on poly(dimethylacrylamide)

Cited by 156 publications

References 3 publications

Role for the Regulator of G-Protein Signaling Homology Domain of G Protein-Coupled Receptor Kinases 5 and 6 in β2-Adrenergic Receptor and Rhodopsin Phosphorylation

Role for the Regulator of G-Protein Signaling Homology Domain of G Protein-Coupled Receptor Kinases 5 and 6 in β2-Adrenergic Receptor and Rhodopsin Phosphorylation

Synthesis and metabolic stability of a tritium‐labelled substance P analogue

Novel Non-PEG Derived Polyethers as Solid Supports. 2. Solid-Phase Synthesis Studies

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