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Peptide modifications differentially alter G protein-coupled receptor internalization and signaling bias.

ABSTRACT: Although G protein-coupled receptors (GPCRs) are targeted by more clinically used drugs than any other type of protein, their ligand development is particularly challenging. Humans have four neuropeptide?Y receptors: hY1R and hY5R are orexigenic, while hY2R and hY4R are anorexigenic, and represent important anti-obesity drug targets. We show for the first time that PEGylation and lipidation, chemical modifications that prolong the plasma half-lives of peptides, confer additional benefits. Both modifications enhance pancreatic polypeptide preference for hY2R/hY4R over hY1R/hY5R. Lipidation biases the ligand towards arrestin recruitment and internalization, whereas PEGylation confers the opposite bias. These effects were independent of the cell system and modified residue. We thus provide novel insights into the mode of action of peptide modifications and open innovative venues for generating peptide agonists with extended therapeutic potential.

SUBMITTER: Made V

PROVIDER: S-EPMC4704863 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Peptide modifications differentially alter G protein-coupled receptor internalization and signaling bias.

Mäde Veronika V Babilon Stefanie S Jolly Navjeet N Wanka Lizzy L Bellmann-Sickert Kathrin K Diaz Gimenez Luis E LE Mörl Karin K Cox Helen M HM Gurevich Vsevolod V VV Beck-Sickinger Annette G AG

Angewandte Chemie (International ed. in English) 20140725 38

Although G protein-coupled receptors (GPCRs) are targeted by more clinically used drugs than any other type of protein, their ligand development is particularly challenging. Humans have four neuropeptide Y receptors: hY1R and hY5R are orexigenic, while hY2R and hY4R are anorexigenic, and represent important anti-obesity drug targets. We show for the first time that PEGylation and lipidation, chemical modifications that prolong the plasma half-lives of peptides, confer additional benefits. Both m ...[more]

PMID: 25065900

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Project description:BackgroundThe human CXC chemokine receptor 2 (CXCR2) is a G protein-coupled receptor (GPCR) interacting with multiple chemokines (i.e., CXC chemokine ligands CXCL1-3 and CXCL5-8). It is involved in inflammatory diseases as well as cancer. Consequently, much effort is put into the identification of CXCR2 targeting drugs. Fundamental research regarding CXCR2 signaling is mainly focused on CXCL8 (IL-8), which is the first and best described high-affinity ligand for CXCR2. Much less is known about CXCR2 activation induced by other chemokines and it remains to be determined to what extent potential ligand bias exists within this signaling system. This insight might be important to unlock new opportunities in therapeutic targeting of CXCR2.MethodsLigand binding was determined in a competition binding assay using labeled CXCL8. Activation of the ELR + chemokine-induced CXCR2 signaling pathways, including G protein activation, β-arrestin1/2 recruitment, and receptor internalization, were quantified using NanoBRET-based techniques. Ligand bias within and between these pathways was subsequently investigated by ligand bias calculations, with CXCL8 as the reference CXCR2 ligand. Statistical significance was tested through a one-way ANOVA followed by Dunnett's multiple comparisons test.ResultsAll chemokines (CXCL1-3 and CXCL5-8) were able to displace CXCL8 from CXCR2 with high affinity and activated the same panel of G protein subtypes (Gαi1, Gαi2, Gαi3, GαoA, GαoB, and Gα15) without any statistically significant ligand bias towards any one type of G protein. Compared to CXCL8, all other chemokines were less potent in β-arrestin1 and -2 recruitment and receptor internalization while equivalently activating G proteins, indicating a G protein activation bias for CXCL1,-2,-3,-5,-6 and CXCL7. Lastly, with CXCL8 used as reference ligand, CXCL2 and CXCL6 showed ligand bias towards β-arrestin1/2 recruitment compared to receptor internalization.ConclusionThis study presents an in-depth analysis of signaling bias upon CXCR2 stimulation by its chemokine ligands. Using CXCL8 as a reference ligand for bias index calculations, no ligand bias was observed between chemokines with respect to activation of separate G proteins subtypes or recruitment of β-arrestin1/2 subtypes, respectively. However, compared to β-arrestin recruitment and receptor internalization, CXCL1-3 and CXCL5-7 were biased towards G protein activation when CXCL8 was used as reference ligand.

Project description:Memantine and ketamine are clinically useful NMDA receptor (NMDAR) open channel blockers that inhibit NMDARs with similar potency and kinetics, but display vastly different clinical profiles. This discrepancy has been hypothesized to result from inhibition by memantine and ketamine of overlapping but distinct NMDAR subpopulations. For example, memantine but not ketamine may inhibit extrasynaptic NMDARs more effectively than synaptic NMDARs. However, the basis for preferential NMDAR inhibition depending on subcellular location has not been investigated systematically. We integrated recordings from heterologously expressed single NMDAR subtypes, kinetic modeling, and recordings of synaptically evoked NMDAR responses in acute brain slices to investigate mechanisms by which channel blockers may distinguish NMDAR subpopulations. We found that memantine and ketamine differentially alter NMDAR desensitization and that memantine stabilizes a Ca2+-dependent desensitized state. As a result, inhibition by memantine of GluN1/2A receptors in tsA201 cells and of native synaptic NMDARs in cortical pyramidal neurons from mice of either sex increased in conditions that enhanced intracellular Ca2+ accumulation. Therefore, differential inhibition by memantine and ketamine based on NMDAR location is likely to result from location dependence of the intensity and duration of NMDAR activation. Modulation of Ca2+-dependent NMDAR desensitization is an unexplored mechanism of inhibitory action with the potential to endow drugs with NMDAR selectivity that leads to superior clinical profiles. Our results suggest that designing compounds to target specific receptor states, rather than specific receptor types, may be a viable strategy for future drug development.SIGNIFICANCE STATEMENT Memantine and ketamine are NMDA receptor (NMDAR) channel-blocking drugs with divergent clinical effects. Understanding mechanistically their differential actions may advance our understanding of nervous system disorders and suggest strategies for the design of more effective drugs. Here, we show that memantine and ketamine have contrasting effects on NMDAR desensitization. Ketamine binding decreases occupancy of desensitized states of the GluN1/2B NMDAR subtype. In contrast, memantine binding increases occupancy of GluN1/2A and native NMDAR desensitized states entered after accumulation of intracellular Ca2+, a novel inhibitory mechanism. These properties may contribute to inhibition of distinct NMDAR subpopulations by memantine and ketamine and help to explain their differential clinical effects. Our results suggest stabilization of Ca2+-dependent desensitized states as a new strategy for pharmaceutical neuroprotection.

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Peptide modifications differentially alter G protein-coupled receptor internalization and signaling bias.

Publications

Peptide modifications differentially alter G protein-coupled receptor internalization and signaling bias.

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