Extracellular loops and ligand binding to a subfamily of Family A G-protein-coupled receptors

Wheatley, Mark; Simms, John; Hawtin, Stuart R.; Wesley, Victoria J; Wootten, Denise; Conner, Matthew T.; Lawson, Zoe; Conner, Alex C.; Baker, Andrew R.; Cashmore, Y; Kendrick, R; Parslow, Rosemary A.

doi:10.1042/bst0350717

Cited by 19 publications

(13 citation statements)

References 27 publications

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“…This region is located at the interface between TM3 and ECL1, and is likely engaged in the formation of a disulphide bond between ECL1 and ECL2. This sequence motif has been shown in a number of other GPCRs to be involved in signal transduction [8,22]. The preservation of the WXFG and DXXCR motifs in GPCRs which have structurally diverse ligands suggests that these motifs are not involved directly in ligand binding.…”

Section: 0 Discussionmentioning

confidence: 99%

Importance of extracellular loop one of the neuropeptide S receptor for biogenesis and function

et al. 2010

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Neuropeptide S (NPS) is the endogenous ligand of a formerly orphan G protein-coupled receptor (GPCR). The NPS receptor (NPSR) belongs to the subfamily of peptide GPCRs and is widely expressed in the brain. NPS promotes arousal and induces anxiolytic-like effects after central administration in rodents. Previously, we have reported that the N107I polymorphism in the human NPS receptor results in a gain-of-function characterized by an increase in agonist potency without changing agonist binding affinity. We have extended our findings by investigating pharmacological and biochemical consequences of mutations in the vicinity of position 107. Alanine substitutions were made for D105 and N101, and stable clones were analyzed for agonist-induced changes of intracellular Ca 2+ . Receptor protein expression was monitored by Western blot and flow cytometry. The mutation D105A produced receptors that have a ∼200-fold higher EC 50 despite elevated total receptor protein and surface expression compared to cell lines expressing the parental receptor NPSR-N107. The mutation N101A resulted in slightly reduced agonist potency without affecting the ability of the protein to form functional receptors. Stable NPSR-A101 clones show little expression of the fully glycosylated form. However, NPSR-A101 receptors are expressed on the cell surface and are functional, suggesting that full glycosylation is not required for receptor function. Our studies suggest that N-linked glycosylation is not important for receptor biogenesis or function, and that residue D105 might be critical for receptor binding.

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Section: 0 Discussionmentioning

confidence: 99%

Importance of extracellular loop one of the neuropeptide S receptor for biogenesis and function

et al. 2010

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“…In particular, in receptors for peptides, glycoprotein hormones, and chemokines, major interaction sites for the endogenous ligands have been described to be present in the extracellular loops and most often in ECL2 (30,68,72,73). Previously, an aromatic residue in position Cϩ4 in other 7TM receptors has been observed to modulate the ligand-induced activation, suggesting a conserved role in the activation mechanism (10).…”

Section: Discussionmentioning

confidence: 99%

Modulation of Constitutive Activity and Signaling Bias of the Ghrelin Receptor by Conformational Constraint in the Second Extracellular Loop

Mokrosiński

Frimurer

Sivertsen

et al. 2012

Journal of Biological Chemistry

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Background: A natural Glu for Ala variant in the ghrelin receptor extracellular loop 2 selectively eliminates constitutive signaling. Results: Computational chemistry and mutational analysis show that charged residues and metal ion sites that induce ␣-helix formation in ECL2 prevent constitutive signaling. Conclusion: Flexibility of ECL2 connecting TM-III and TM-V is essential for spontaneous receptor signaling. Significance: Clarification of ECL2 structural constraint is important for receptor signaling.

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“…Intriguingly, the extracellular loop of rhodopsin is also believed to move during light activation of this GPCR, and the signaling role of extracellular loops in other Class A GPCRs is just starting to be revealed as well (55). Ste2p is a Class D GPCR and rhodopsin is a Class A GPCR.…”

Section: Discussionmentioning

confidence: 99%

Cross-Linking of a DOPA-Containing Peptide Ligand into Its G Protein-Coupled Receptor

et al. 2009

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The interaction between a 3,4-dihydroxylphenylalanine (DOPA) labeled analog of the tridecapeptide α-factor (W-H-W-L-Q-L-K-P-G-Q-P-M-Y) and Ste2p, a Saccharomyces cerevisiae model G protein-coupled receptor (GPCR), has been analyzed by periodate-mediated cross-linking. Chemically synthesized α-factor with DOPA substituting for tyrosine at position 13 and biotin tagged onto lysine 7 ([Lys 7 (BioACA),-Nle 12 ,DOPA 13 ]α-factor; Bio-DOPA-α-factor) was used for crosslinking into Ste2p. The biological activity of Bio-DOPA-α-factor was about one-third that of native α-factor as determined by growth arrest assay and exhibited about a ten-fold lower binding affinity to Ste2p. Bio-DOPA-α-factor cross-linked into Ste2p as demonstrated by western blot analysis using a Neutravidin-HRP conjugate to detect Bio-DOPA-α-factor. Cross-linking was inhibited by excess native α-factor and an α-factor antagonist. The Ste2p-ligand complex was purified using a metal ion affinity column, and after cyanogen bromide treatment, avidin affinity purification was used to capture Bio-DOPA-α-factor-Ste2p cross-linked peptides. MALDI-TOF spectrometric analyses of the cross-linked fragments showed that Bio-DOPA-α-factor reacted with the Phe 55 -Met 69 region of Ste2p. Cross-linking of Bio-DOPA-α-factor was reduced by 80% using a cysteine-less Ste2p (Cys59Ser). These results suggest an interaction between position thirteen of α-factor and residue Cys 59 of Ste2p. This study is the first to report DOPA cross-linking of a peptide hormone to a GPCR and the first to identify a residue-to-residue cross-link between Ste2p and α-factor thereby defining a specific contact point between the bound ligand and its receptor.G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins associated with signaling systems present in mammalians, plants, protozoans, fungi, and metazoans (1). Malfunctions of GPCRs contribute to diseases such as Alzheimer's, Parkinson's disease, diabetes, color blindness, asthma, depression, hypertension, stress, cardiovascular, and immune disorders (2,3). All GPCRs share a common structure consisting of seven transmembrane domains (TMDs) connected by three extracellular and three intracellular loops (1,4,5). Upon binding of their ligands, GPCRs undergo conformational changes that transduce NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 March 10. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the signal into the cell by activating a cascade of protein-protein interactions initiated through a heterotrimeric G protein complex (6).Ste2p, the Saccharomyces cerevisiae α-factor pheromone receptor, has been studied as a model for peptide-responsive GPCRs (7,8). Though Ste2p does not share recognizable sequence similarity with mammalian GPCRs or even with Ste3p, the a-factor pheromone receptor of yeast, all GPCRs have strong structural and functional similarities (9,10). For example, the packing and interactions between the fifth and sixth tr...

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Extracellular loops and ligand binding to a subfamily of Family A G-protein-coupled receptors

Cited by 19 publications

References 27 publications

Importance of extracellular loop one of the neuropeptide S receptor for biogenesis and function

Importance of extracellular loop one of the neuropeptide S receptor for biogenesis and function

Modulation of Constitutive Activity and Signaling Bias of the Ghrelin Receptor by Conformational Constraint in the Second Extracellular Loop

Cross-Linking of a DOPA-Containing Peptide Ligand into Its G Protein-Coupled Receptor

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