Site-Specific Epitope Tagging of G Protein-Coupled Receptors by Bioorthogonal Modification of a Genetically Encoded Unnatural Amino Acid

Biochemistry ◽  
2013 ◽  
Vol 52 (6) ◽  
pp. 1028-1036 ◽  
Author(s):  
Saranga Naganathan ◽  
Shixin Ye ◽  
Thomas P. Sakmar ◽  
Thomas Huber
Keyword(s):  
Amino Acid ◽  
G Protein ◽  
Unnatural Amino Acid ◽  
G Protein Coupled Receptors ◽  
Epitope Tagging ◽  
Site Specific ◽  
Specific Epitope ◽  
G Protein Coupled

scholarly journals Site-specific Incorporation of Keto Amino Acids into Functional G Protein-coupled Receptors Using Unnatural Amino Acid Mutagenesis

2007 ◽  
Vol 283 (3) ◽  
pp. 1525-1533 ◽  
Author(s):  
Shixin Ye ◽  
Caroline Köhrer ◽  
Thomas Huber ◽  
Manija Kazmi ◽  
Pallavi Sachdev ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
G Protein ◽  
Unnatural Amino Acids ◽  
Unnatural Amino Acid ◽  
G Protein Coupled Receptors ◽  
Calcium Flux ◽  
Unnatural Amino Acid Mutagenesis ◽  
Amino Acid Mutagenesis ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-l-phenylalanine (Acp) and p-benzoyl-l-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to ∼50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5–2 μg/107 cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors.

scholarly journals Minireview: Nutrient Sensing by G Protein-Coupled Receptors

2013 ◽  
Vol 27 (8) ◽  
pp. 1188-1197 ◽  
Author(s):  
Eric M. Wauson ◽  
Andrés Lorente-Rodríguez ◽  
Melanie H. Cobb
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
G Protein ◽  
Taste Receptor ◽  
Sweet Taste ◽  
Nutrient Sensing ◽  
G Protein Coupled Receptors ◽  
Amino Acid Availability ◽  
Class C ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) are membrane proteins that recognize molecules in the extracellular milieu and transmit signals inside cells to regulate their behaviors. Ligands for many GPCRs are hormones or neurotransmitters that direct coordinated, stereotyped adaptive responses. Ligands for other GPCRs provide information to cells about the extracellular environment. Such information facilitates context-specific decision making that may be cell autonomous. Among ligands that are important for cellular decisions are amino acids, required for continued protein synthesis, as metabolic starting materials and energy sources. Amino acids are detected by a number of class C GPCRs. One cluster of amino acid-sensing class C GPCRs includes umami and sweet taste receptors, GPRC6A, and the calcium-sensing receptor. We have recently found that the umami taste receptor heterodimer T1R1/T1R3 is a sensor of amino acid availability that regulates the activity of the mammalian target of rapamycin. This review focuses on an array of findings on sensing amino acids and sweet molecules outside of neurons by this cluster of class C GPCRs and some of the physiologic processes regulated by them.

Characteristic amino acid combinations in olfactory G protein-coupled receptors

2007 ◽  
Vol 67 (1) ◽  
pp. 154-166
Author(s):  
Elena V. Samsonova ◽  
Peter Krause ◽  
Thomas Bäck ◽  
Ad P. IJzerman
Keyword(s):  
Amino Acid ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
G Protein Coupled

scholarly journals GPCRtm: An amino acid substitution matrix for the transmembrane region of class A G Protein-Coupled Receptors

2015 ◽  
Vol 16 (1) ◽  
Author(s):  
Santiago Rios ◽  
Marta F. Fernandez ◽  
Gianluigi Caltabiano ◽  
Mercedes Campillo ◽  
Leonardo Pardo ◽  
...  
Keyword(s):  
Amino Acid ◽  
G Protein ◽  
Amino Acid Substitution ◽  
G Protein Coupled Receptors ◽  
Substitution Matrix ◽  
Transmembrane Region ◽  
Class A ◽  
Amino Acid Substitution Matrix ◽  
G Protein Coupled

scholarly journals IMMUNOLOGICAL INVESTIGATIONS OF HHV-6 U12 AND U51 ENCODED PROTEINS INVOLVEMENT IN AUTOIMMUNE THYROIDITIS DEVELOPMENT

2020 ◽  
Author(s):  
Alina Sultanova ◽  
Maksims Cistjakovs ◽  
Liba Sokolovska ◽  
Egils Cunskis ◽  
Modra Murovska
Keyword(s):  
Amino Acid ◽  
G Protein ◽  
Autoimmune Thyroiditis ◽  
Synthetic Peptides ◽  
Human Herpesvirus ◽  
Amino Acid Sequences ◽  
G Protein Coupled Receptors ◽  
Igm Antibodies ◽  
Specific Igg ◽  
G Protein Coupled

AbstractHuman herpesvirus 6 (HHV-6) is a human pathogen with a wide cell tropism and many immunomodulating properties. HHV-6 has been linked to the development of multiple diseases, among them – autoimmune. Conflicting evidence implicates HHV-6 in autoimmune thyroiditis (AIT). HHV-6 contains two genes (U12 and U51) that encode putative homologues of human G-protein-coupled receptors (GPCR) like CCR1, CCR3 and CCR5. It has been shown that proteins encoded by HHV-6 U12 and U51 genes can be expressed on the surface of epithelial and some peripheral blood mononuclear cells populations, which makes them a potential cause for evoking autoimmunity.The aim of this study was to identify potentially immunogenic synthetic peptides derived from HHV-6 U12 and U51 amino acid sequences and to find evidences of the possible involvement of these proteins in AIT development. 62 AIT patients positive for HHV-6 infection were enrolled in this study. 30 different synthetic peptides designed from HHV-6 U12 and U51 proteins’ amino acid sequences, as well as, recombinant human CCR1, CCR3 and CCR5 proteins were used for suspension multiplex immunological assay (SMIA) to detect specific IgG, and IgM antibodies.HHV-6 peptide specific IgG and IgM antibodies were found in patient’s samples, with higher signals for IgM antibodies, which is indicative of reactivation and active HHV-6 infection. As well recombinant CCR1 and CCR5 showed high signals on IgM antibodies which is indicating on the presence of potential auto-antibodies against human G protein-coupled receptors. No cross reactivity between HHV-6 peptide specific antibodies and human recombinant CCR1, CCR3 and CCR5 was found, however, the possibility of cross-reactive autoantibodies specific for structural epitopes cannot be excluded.

scholarly journals Cargo regulates clathrin-coated pit invagination via clathrin light chain phosphorylation

2018 ◽  
Vol 217 (12) ◽  
pp. 4253-4266 ◽  
Author(s):  
Hannes Maib ◽  
Filipe Ferreira ◽  
Stéphane Vassilopoulos ◽  
Elizabeth Smythe
Keyword(s):  
G Protein ◽  
Light Chain ◽  
Transferrin Receptor ◽  
G Protein Coupled Receptors ◽  
Light Chains ◽  
Coated Pits ◽  
Selective Uptake ◽  
Site Specific ◽  
Specific Manner ◽  
G Protein Coupled

Clathrin light chains (CLCs) control selective uptake of a range of G protein–coupled receptors (GPCRs), although the mechanism by which this occurs has remained elusive thus far. In particular, site-specific phosphorylation of CLCb controls the uptake of the purinergic GPCR P2Y12, but it is dispensable for the constitutive uptake of the transferrin receptor (TfR). We demonstrate that phosphorylation of CLCb is required for the maturation of clathrin-coated pits (CCPs) through the transition of flat lattices into invaginated buds. This transition is dependent on efficient clathrin exchange regulated by CLCb phosphorylation and mediated through auxilin. Strikingly, this rearrangement is required for the uptake of P2Y12 but not TfR. These findings link auxilin-mediated clathrin exchange to early stages of CCP invagination in a cargo-specific manner. This supports a model in which CCPs invaginate with variable modes of curvature depending on the cargo they incorporate.

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